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ISSN 1560-7259 (print edition)
TURCZANINOWIA
ISSN 1560-7267 (online edition)
УДК 582.394.7:581.4(59)+575.86
Comprehensive analysis of relationships of the representatives of subfamily
Cryptogrammoideae (Pteridaceae)
A. V. Vaganov1, 3 5*, I. I. Gureyeva2, 6, M. V. Skaptsov1, 7, A. A. Kuznetsov2, 8, R. S. Romanets2, 9, A. V. Salokhin4, 10, M. G. Kutsev1, 11, A. I. Shmakov1, 12
1 Altai State University, Lenina Pr., 61, Barnaul, 656049, Russian Federation
2 Tomsk State University, Lenina Pr., 36, Tomsk, 634050, Russian Federation
3 Sakhalin Branch of the Botanical Garden Institute FEB RAS, Gorkogo St., 25, Yuzhno-Sakhalinsk, 693023, Russian Federation 4Botanical Garden-Institute FEB RAS, Makovskogo St., 142, Vladivostok, 690024, Russian Federation 5E-mail: vaganov_vav@mail.ru; ORCID iD: https://orcid.org/0000-0002-7584-5150 6E-mail:gureyeva@yandex.ru; ORCID iD: https://orcid.org/0000-0002-5397-6133 7 E-mail: mr.skaptsov@mail.ru; ORCID iD: https://orcid.org/0000-0002-4884-0768 8E-mail:ys.tsu@mail.ru; ORCID iD: https://orcid.org/0000-0002-5553-2958 9E-mail: romanromanets4990@gmail.com; ORCID iD: https://orcid.org/0000-0003-3934-6079 10E-mail: al-xv@mail.ru; ORCID iD: https://orcid.org/0000-0002-4902-8472 11 E-mail: m_kucev@mail.ru; ORCID iD: https://orcid.org/0000-0003-2284-6851 12E-mail: alex_shmakov@mail.ru; ORCID iD: https://orcid.org/0000-0002-1052-4575
*Corresponding author
Keywords: Coniogramme, Cryptogramma, Cryptogrammoideae, ferns, Llavea, phylogeny, phylogeography, Pteridaceae, scanning electronic microscopy (SEM), spore morphology, systematics.
Summary. This research is the first comprehensive analysis of the intrageneric relationships inside the subfamily Cryptogrammoideae: 14 taxa of Coniogramme and one species of Cryptogramma were involved additionally in the molecular phylogenetic studies based on rbcL gene of plastid DNA; spore morphology of 32 taxa of cryptogrammoid ferns, namely 22 taxa of Coniogramme, nine species of Cryptogramma and one species of Llavea were studied using scanning electronic microscopy (SEM); 31 taxon of Cryptogrammoideae were studied using herbarium data from Herbaria across Europe and Asia (P, PE, LE, VLA, ALTB, TK) according to global botanical and geographical zones. As a result of this comprehensive analysis, we established a deep divergence of Coniogramme merillii in Coniogramme superclade: this species is the sister lineage to the remainder of Coniogramme. We revealed also the separateness of Co. suprapilosa from Co. rosthornii and Co. longissima, Co. africana from Co. lanceolata and Co. fraxinea, Co. robusta from Co. jinggangshanensis, Co. wilsonii and Co. japonica. Among Cryptogramma species, the relationship of Far Eastern Cr. gorovoi with Cr. crispa from the Caucasus and the Turkish endemic Cr. bithynica but not with any Far Eastern species was revealed. Spores of Coniogramme are characterized by simple smooth, granulate and papillate macro-ornamentation, spores of Cryptogramma species have the more coarse colliculate or tuberculate macro-ornamentation. Peculiarities of macro-ornamentation allow us to define six spore types in cryptogrammoid ferns: four spore types in Coniogramme and two spore types in Cryptogramma; the same spore type we assigned for Llavea cordifolia and Coniogramme suprapilosa. In Coniogramme, the grouping of species attending the spore type does not agree with existing classification and phylogenetic hypotheses. Genetic separateness of Co. suprapilosa corresponds with its exceptional verrucate spore sculpture not found in other Coniogramme species. In Cryptogramma, the grouping on the spore types corresponds with other morphological characteristics, existing system and molecular phylogeny. Spore ornamentation has diagnostic value in the recognition of cryptogrammoid taxa at the generic and section (in Cryptogramma) level.
Поступило в редакцию 31.04.2023 Принято к публикации 29.06.2023
Submitted 31.04.2023 Accepted 29.06.2023
Sino-Japanese and Sino-Himalaian regions of Eastern Asiatic Subkingdom are the centers of origin and diversity for subfamily Cryptogrammoideae and especially for the genus Coniogramme.
Комплексный анализ родства представителей подсемейства Cryptogrammoideae (Pteridaceae)
А. В. Ваганов1' 3, И. И. Гуреева2, М. В. Скапцов1, А. А. Кузнецов2, Р. С. Романец2, А. В. Салохин4, М. Г. Куцев1, А. И. Шмаков1
1 Алтайский государственный университет, пр. Ленина, д. 61, г. Барнаул, 656049, Россия 2 Томский государственный университет, пр. Ленина, д. 36, г. Томск, 634050, Россия 3 Сахалинский филиал Института ботанического сада ДВО РАН, ул. Горького, д. 25, г. Южно-Сахалинск, 693023, Россия 4 Ботанический сад-институт ДВО РАН, ул. Маковского, д. 142, г. Владивосток, 690024, Россия
Ключевые слова: морфология спор, папоротники, систематика, сканирующая электронная микроскопия (СЭМ), филогения, филогеография, Coniogramme, Cryptogramma, Cryptogrammoideae, Llavea, Pteridaceae.
Аннотация. Представлен первый комплексный анализ родственных отношений в подсемействе Cryptogrammoideae: 14 таксонов Coniogramme и один вид Cryptogramma были дополнительно привлечены к молеку-лярно-филогенетическим исследованиям на основе гена rbcL пластидной ДНК; с применением метода сканирующей электронной микроскопии (СЭМ) изучены споры 32 таксонов криптограммоидных папоротников, выявлено распространение 31 таксона Cryptogrammoideae в соответствии с глобальным ботанико-географи-ческим районированием с использованием данных из Гербариев Европы и Азии (P, PE, LE, VLA, ALTB, TK). В результате установлена глубокая дивергенция Coniogramme merillii в суперкладе Coniogramme: этот вид является родственной линией остальной части рода; выявлена обособленность Co. suprapilosa от Co. rosthornii и Co. longissima, Co. africana от Co. lanceolata и Co. fraxinea, Co. robusta от Co. jinggangshanensis, Co. wilsonii и Co. japonica. Среди видов Cryptogramma выявлено родство дальневосточного Cr. gorovoi с кавказской и европейской Cr. crispa и турецким эндемиком Cr. bithynica, а не с каким-либо из дальневосточных видов. Споры Coniogramme характеризуются простой гладкой, зернистой и папиллятной макроскульптурой, споры видов Cryptogramma имеют более грубую колликулятную или бугорчатую макроскульптуру. Особенности макроскульптуры позволяют выделить шесть типов спор у криптограммоидных папоротников: четыре типа спор у Coniogramme и два у Cryptogramma; один и тот же тип спор выявлен у Llavea cordifolia и Coniogramme suprapilosa. Для рода Coniogramme группировка видов по типу спор не согласуется с существующей классификацией и филогенетическими гипотезами. Генетическая обособленность Co. suprapilosa соотносится с необнаруженной у других видов Coniogramme бородавчатой скульптурой спор. В роде Cryptogramma группировка видов по типу спор согласуется с морфологическим характеристиками, существующей системой и молекулярной филогенией. Скульптура спор имеет диагностическое значение при распознавании таксонов криптограммоидных папоротников на родовом и секционном (в роде Cryptogramma) уровне. Центрами происхождения и разнообразия подсемейства Cryptogrammoideae, особенно рода Coniogramme, являются китайско-японский и китайско-гималайский регионы Восточноазиатского подцарства.
Introduction
According to the modern system of the family Pteridaceae E. D. M. Kirchn, three genera - Coniogramme Fée, Cryptogramma R. Brown, and Llavea Lag. - belong to the subfamily Cryptogrammoideae S. Lindsay (Smith et al., 2006; Prado et al., 2007; Schuettpelz et al., 2007; Christenhusz et al., 2011; Zhang et al., 2013; PPG I, 2016). The separate family Cryptogrammaceae Pic. Serm. was proposed by R. E. G. Pichi-Sermolli (1963) who included three genera:
Cryptogramma, Llavea and Onychium Kaulfuss. These representatives have similar morphological features of sporophytes, such as the structure of the terminal lobes of the fertile fronds, the presence of indusia, and partial to complete frond dimorphism. R. Ch. Ching (1940, 1978) did not accept Pichi-Ser-molli's Cryptogrammaceae and put Cryptogramma, Llavea, and Onychium into the family Sinopterida-ceae Koidzumi. R. M. Tryon and A. F. Tryon (1982) included these three genera as part of tribe Chei-lantheae within the large family Pteridaceae. Later,
R. M. Tryon et al. (1990) transferred Onychium into the subfamily Taenitidoideae of Pteridaceae, whereas Ctyptogramma and Llavea remained in the subfamily Cheilanthoideae.
Molecular phylogenetic study based on the plas-tid DNA loci rbcL, atpB, and atpA confirmed indisputable relationship of three genera Cryptogramma, Llavea, and Coniogramme into the large family Pteridaceae, where the topology of "cryptogrammoid-clade" is well-supported and clade is monophyletic (Zhang et al., 2005; Schuettpelz et al., 2007; PPG I, 2016). A relationship between Coniogramme, Cryptogramma, and Llavea was never suggested based on morphology alone. This group is highly variable in morphology, and a clear morphological synapo-morphy is lacking (Schuettpelz et al., 2007). Analysis of six-locus plastid dataset (rbcL, rbcL-accD, rbcL-atpB, rps4-trnS, trnG-trnR, and trnP-petG) demonstrated monophyly of Coniogramme and Cryptogramma within Llavea/Coniogramme/Cryptogramma clade; their relationship as sister taxa was not suggested until the advent of molecular research (Schuettpelz et al., 2007; Metzgar et al., 2013). According to a molecular study by J. S. Metzgar et al. (2013), Coniogramme and Cryptogramma are more closely related to each other than to Llavea, i. e. Lla-vea is sister to Coniogramme and Cryptogramma together.
Coniogramme is a largest genus in the subfamily, numbering from 20 (PPG I, 2016) or 25-30 (Zhang, Ranker, 2013) to 50 (Wang et al., 2020) species. This paleotropical genus is mainly distributed in the tropical and subtropical regions of Asia and extends to Africa and Malesia from Malaya and Sumatra east to New Guinea and in the Pacific to Samoa and Hawaiian Islands (Hieronymus, 1916; Tryon, Lugardon, 1991; Zhang, Ranker, 2013). A half of the representatives of the genus Coniogramme is endemic species. The genus is one of the most complex in the family Pteridaceae due to the high polymorphism of morphological features and hybridogenicity. The reason for a large number of synonyms among described taxa is the lack of clear diagnostic features for their identification. The most valuable diagnostic features are the shape of the frond lobes and their margins, the presence and type of hydathodes, the colour of the stipe and rachis (Zhang, Ranker, 2013). Recently a new feature was revealed for representatives from series Coniogramme - the number of annulus cells, which is stable at the species level and has great phy-logenetic significance (Wang et al., 2020). Based on the morphology of sporophyte, the genus Conio-gramme was divided into the section Notogramme
(C. Presl) Ching ex K. H. Shing and section Coniogramme with series Coniogramme and Serratae Ching ex K. H. Shing (Shing, 1981). Twelve Coniogramme species were included in the phylogenetic reconstruction based on five chloroplast markers (matK, psbA-trnH, rbcL, rps4-trnS, trnL-F) by Wang et al. (2020). According to this study, Coniogramme ser. Coniogramme as formally distinguished by the morphology of pinnule margins is not a monophy-letic group.
Among Coniogramme species, diploids (n = 30) Coniogramme affinis Hieron. and Co. procera Fée (Khullar et al., 1988; Kato et al., 1992), tetraploids (n = 60) Co. africana Hieron. and Co. rosthorni Hieron. (Löve et al., 1977) and hexaploid (n = 90) Co. pilosa Hieron. (Löve et al., 1977) were identified. Two ploidy levels (n = 30, 60) were indicated for Co. fraxinea (D. Don) Diels, Co. japonica (Thunb.) Diels, and Co. intermedia Hieron. (Kurita, 1972; Singh, Roy, 1988; Weng, Qiu, 1988; Kato et al., 1992).
The genus Cryptogramma includes about ten (Al-verson, 1989a, b; Vaganov et al., 2010; Zhang, Ranker, 2013) extratropical species distributed mainly in the northern hemisphere, one species - Cr. fumari-ifolia (Phil. ex Baker) H. Christ - occurs in the south of South America (Raven, 1963; Alverson, 1989a; Zhang G. M., Zhang X. Ch., 2003; Metzgar et al., 2013). The genus includes two sections, Cryptogram-ma and Homopteris Rupr., sometimes considered as subgenera Cryptogramma and Homopteris (Rupr.) Tzvelev (Tzvelev, 1989; Shmakov, 2001). Species from the sect. Cryptogramma have erect rhizomes, fronds with a more coriaceous texture and generally prefer acidic, rocky habitats (Alverson, 1989a). Cryptogramma stelleri (S. G. Gmel.) Prantl, the only species of the sect. Homopteris (or subgen. Homopteris), has creeping rhizome, membraneous texture of fronds, and is often calciphilous (Hultén, Fries, 1986; Alverson, 1993). Based on molecular-phylo-genetic studies, J. S. Metzgar et al. (2013, 2016) supported the recognition of nine taxa in Cryptogram-ma at the species level, as well as the monophyly of the two sections Cryptogramma and Homopteris.
The following chromosome counts are known for Cryptogramma: n = 30 (diploids) in Cr. acrostichoi-des R. Br., Cr. brunoniana Wall. ex Hook. et Grev., Cr. cascadensis Alverson, Cr. stelleri (Khullar et al., 1988; Alverson, 1989a; Gervais et al., 1999) and n = 60 (tetraploids) in Cr. crispa (L.) R. Br. and Cr. sit-chensis (Rupr.) T. Moore (Alverson, 1989a; Pajaron et al., 1999).
Llavea is a monotypic American genus with a single species, Llavea cordifolia Lag., distributed in
the Central America from Mexico to Guatemala and Costa Rica (Palacios-Rios et al., 2017a). Llavea cordi-folia is diploid with n = 29 (Knobloch, 1967).
Today, many studies have shown that characters of spore ornamentation, identified by scanning electron microscopy (SEM), are of diagnostic value and in most cases are congruent with the results of molecular phylogenetic studies (Gureyeva, Kuznetsov, 2015; Palacios-Rios et al., 2017b; Chao, Huang, 2018; Vaganov et al., 2020; Irfan et al., 2021; Vagan-ov, 2022). To date, there is some information on the spore morphology of Cryptogramma, Coniogramme, and Llavea species in the form of short descriptions of morphology and/or SEM-photographs (Tryon, Lugardon, 1991; Yu et al., 2001; Zhang G. M., Zhang X. Ch., 2003; Gureyeva et al., 2009; Vaganov et al., 2010, 2011; Vaganov, 2016).
Molecular phylogenetis study based on six plas-tid DNA regions (rbcL, rbcL-accD, rbcL-atpB, rps4-trnS, trnG-trnR, and trnP-petG) of nine species Cryptogramma was performed by J. S. Metzgar et al. (2013, 2016). This study supported the recognition of nine published Cryptogramma taxa at the species level, most species were found to be reciprocally monophyletic. Within Coniogramme, methods of molecular phylogenetic analysis based on five plas-tid DNA regions (matK, psbA-trnH, rbcL, rps4-trnS,
and trnL-F) were applied for 11 species for species formally grouped into a series Coniogramme (Wang et al., 2020). This study has indicated that the present defined Coniogramme series Coniogramme is not a monophyletic group, and C. merrillii is a sister to the rest of Coniogramme.
The aim of presented work is an integrating study of the genera Coniogramme, Cryptogramma, and Llavea (Cryptogrammoideae, Pteridaceae) including molecular phylogenetic analysis, morphology of spores and phylogeography based on large number of taxa, including taxa that were not previously involved in the studies.
2. Material and methods
Information on the distribution of Coniogramme, Cryptogramma, and Llavea taxa was compiled from Herbaria across Europe and Asia (P, PE, LE, VLA, ALTB, and TK), as well as from Global Biodiversity Information Facility (GBIF, 2023) aggregated data of digitized herbarium specimen. Google Earth was used to clarify coordinates from the herbarium labels. The QGIS3.14 software was used to map the species distribution. In the botanical-geographical zoning of the Earth, we followed the global vegetation classification by R. V. Kamelin (2017).
Table 1
Studied taxa, herbarium number and place of storage of the examined specimens (an asterisks mark the taxa which spores were studied for the first time)
No. Taxon Country, herbarium number, and herbarium code
1 *Coniogramme affinis (Wall.) Hieron. China, Sichuan. Song Zipu. 01 VIII 1954. PE-00587617 (PE)
2 Co. africana Hieron. Cameroon, Southwest region, J.-M. Onana. VII 2001. P00968223 (P)
3 *Co. caudiformis Ching et K. H. Shing China, Hunan. Xi Jian-Ming. 08 XI 2007. PE-01769269 (PE)
4 *Co. emeiensis Ching et K. H. Shing China, Hunan, Liu Bingrong, Yan Yuehong. 26 VI 2007. PE-01769317; China, Nanchuan, Li Guofeng, 30 VII 1957, PE-00598529; China, Guangxi, Qin Renchang, 13 VIII 1928, PE-00598215; China, Sichuan, Emai Shan, R.C. Ching, PE-00598262, type. (PE)
5 *Co. falcipinna Ching et K. H. Shing China, Sangzhi, Beijing Team, PE-01570623 (PE)
6 Co. fraxinea (Don) Diels China, Kweichow, A. N. Steward, C. Y. Chiao, H. C. Cheo. 16 IX 1931 (LE); China, Yunnan, Sino-Soviet Mission, PE-00587823; China, Yunnan, Yunnan University, 12 X 1965, PE-00587775 (PE)
7 Co. intermedia Hieron. var. intermedia China, Hunan, Liu Linhan. 25 X 1962. PE-00587987; China, Yunnan, Chung Ju-shi, 26 IX 1958 (LE); Japan, Kyushu, M. Kawabata, 26 VII 1963. PE-01715052; Japan, R. Itoo. PE-01715045; China, Yunnan, Wang Zhongren. 26 I 1978. PE-00598219; Japan, Shimonada-machi. Y. Miyoshi. 23 IX 1956. PE-01715073; China, Hubei. Wuhan University. 17 VIII 1951. PE-00587665; China, Sichuan. Xing Gongxia, Lang Kaiyong. 24 VIII 1963. PE-00598241 (PE)
Table 1 (continued)
No. Taxon Country, herbarium number, and herbarium code
8 *Co. intermedia var. glabra Ching China, Sichuan, Xing Gongxia, Lang Kaiyong. 19 VIII 1963. PE-00598501; China, Sichuan, Xing Gongxia, Lang Kaiyong. 16 VIII 1963. PE-00598493; China, Tibet, T. Naito et al. 14 V 1986. PE-00587774 (PE)
9 Co. japonica (Thunb.) Diels Japan, Yokosuka. M. Le D Sayatier. 1866. № 1572 (LE); China, Henan, Plant Resources Investigation Team. 09 VII 1984. PE-00598017; China, Jiangxi, Cheng Jingfu. 22 VI 1973. PE-00587754; China, Guangdong. Deng Liang. 01 IX 1958. PE-00587773; China, Anhui, M. B. Deng. 18 X 1996. PE-01644009; China, Hubei, Dai Lunying, Qian Chonghai. 19 X 1951. PE-00587832 (PE)
10 *Co. lanceolata Ching China, Yunnan, R. C. Ching, № 50448. 24 XI 1952. PE-00598191 (PE)
11 *Co. madagascariensis C. Chr. Madagaskar, Antsiranana, F. Rakotondrainibe. 3 XI 1994. P00006493(P)
12 *Co. petelotii Tardieu China, Yunnan, T. T. Yu. 21 IX 1938. PE-00598322 (PE)
13 Co. pilosa Hieron. U. S. A., A. A. Heller. IX 1895. P00518737 (P)
14 *Co. procera (Wall.) Fée China, Yunnan, K. M. Feng. PE-00598332 (PE)
15 *Co. pubescens Hieron. India, Simpla, Zhang Xianchun. 16 IX 2001. PE-01715075 (PE)
16 *Co. robusta Christ var. robusta China, Hunan, Beijing Team. 27 VIII 1988. PE-01557759; China, Hunan, Wu Shifu et al. 07 IX 1989. PE-00598372; China, Guizhou, Liu Zhengyu. 29 VII 1996. PE-00001394 (PE)
17 *Co. robusta var. splendens Ching et K. H. Shing China, Hunan, Zhou Joy, Ouyang Haibo, 22 VIII 2007. PE-01769305 (PE)
18 *Co. rosthorni Hieron. China, Taibai, Wang Fayan, Fu Kunjun, Huang Boxing, etc. 08 X 1955. PE-00598472 (PE)
19 *Co. rubicaulis Ching China, Guangxi. 17 IX 1989. PE-02019193 (PE)
20 *Co. serrulata Fée China, Yunnan. 09 V 1913. PE-01715042; China, Gongshan, Jin Xh., Wang L.S., Wang Q. et al. 20 VII 2013. PE-02004117 (PE)
21 *Co. suprapilosa Ching China, Sichuan, Kong Xianxu. 30 VII 1978. PE-00598461 (PE)
22 *Co. wilsonii Hieron. China, Wang Tso-Pin. PE-00598522; China, Henan. 1959. PE-00598513 (PE)
23 Cryptogramma acrostichoides R. Br. U. S. A., Alaska, 29 VII 1990, S. Kharkevich (VLA); Russia, Kurile Islands, V. Yu. Barkalov. 14 XII 1985 (LE)
24 Cr. brunoniana Wall. ex Hook. et Grev. China, Sikkim, J. J. (LE); China, Shaanxi, Girald, PE-00576078; China, Chongqing, Eighth Forest Manager Brigade. 25 VII 1958. PE-00576079 (PE)
25 Cr. cascadensis E.R. Alverson U. S. A., California, A. A. Heller. 27 VII 1903 (LE); U. S. A., Geo. Hansen. 10 I 1894. P01270635 (P)
26 Cr. crispa (L.) R. Br. Russia, Murmansk, M. Kachurin. VIII 1937. (LE); France, Valsenestre, R. Barbezat. 27 VIII 1939. P01572968 (P)
27 *Cr. fumariifolia (Phil.) Christ South America, P01323621 (P)
28 Cr. gorovoii Vaganov et Shmakov Russia, Sakhalin, Pavlova and Pankov. 13VIII 1966. ALTB1010000027, typus (ALTB)
29 Cr. raddeana Fomin Russia, Buryatia, Lake Baikal. Baikal ridge. T.V. Egorova and V. N. Siplivinsky, 1-3 VIII 1967 (LE)
30 Cr. sitchensis (Rupr.) T. Moore Russia, Magadan, V. A. Golub. 08 XII 1933. No. 8 (LE); U. S. A., Alaska, C. Kharkevich. 22 VII 1990 (VLA)
31 Cr. stelleri (S.G. Gmel.) Prantl Russia, Altai, A. Pyak. 25 VII 1990. TK-004507 (TK)
32 Llavea cordifolia Lag. Mexico, Nuevo Leon, C. G. Pringle. 12 III 1906 (LE)
Morphology and morphometry of spores
Samples for studying spore morphology using scanning electron microscopy (SEM) were collected from herbarium specimens (P, PE, ALTB, LE, TK, and VLA), including type material of Coniogramme emeiensis Ching et K.H. Shing and Cryptogramma gorovoii Vaganov et Shmakov. Fifty-six samples comprising 32 taxa, including 22 taxa of Coniogramme, nine species of Cryptogramma, and one species of Llavea were analyzed (Table 1). Spores of 19 taxa were studied for the first time. Identification of species was confirmed by the first author (AVV).
Ultrastructure of spores was performed using SEM, only mature spores were used for observations. Spores were mounted on metal stubs with adhesive double-sided tape, covered with gold or gold/ palladium mixture under vacuum, and examined with Mini-SEM SNE-4500M (SEC Co. Ltd, Korea) at Tomsk State University (Tomsk, Russia) and Hitachi S 3400 N (Hitachi Science Systems, Ltd, Japan) at the Institute for Water and Environmental Problems of Siberian Branch of Russian Academy of Science (Barnaul, Russia). Spore surfaces were scanned in a high vacuum at voltage of 25 or 30 kV, with 400x to 14000x magnification.
SEM-micrographs of spores in the polar (distal and proximal) and in equatorial positions were used for the description of the shape and ornamentation of spores. The shape of the distal and proximal parts of the spore as seen in the equatorial position we described using the terms of Nayar and Devi (1966). In terminology for the description of spore ornamentation, we followed basically Lellinger (2002). Here we used the following terms for description of ornamentation (sculpture) of spores in cryptogrammoid ferns:
- smooth: the surface is smooth without sculptural elements visible in SEM;
- granulate: the presence of minute, granule-like elements;
- papillate: the presence of minute densely spaced papillae;
- verrucate: the presence of elements that are slightly longer than wide, irregular in shape (wartlike), and have obtuse to round apex; verrucae are formed by inner massive consolidated layer of perispore;
- colliculate: the presence of elements that are contiguous, broadly attached, no higher than wide, and with an obtuse to round apex;
- tuberculate: the presence of tubercles that are as long as wide and have a rarely truncate or an obtuse to round apex.
SEM-micrographs of spores in distal, proximal and equatorial positions were used for the measurements using the software "Image J". A total of 1863 spore images were analyzed in detail. Equatorial diameter (ED) in polar view, length (LL) and width (LW) of laesura arms in proximal view were used as the main biometrical characters of spores. All measurements were taken on 25 spores from each species. Datasets were processed using the software Statistica 8.0 (2008, http://www.statsoft. com) with estimation of the mean (M) and standard error (SE). The minimum, mean, and maximum are given in micrometres.
Molecular phylogenetic analyses
Sequences for rbcL gene of plastid DNA were used for phylogenetic analyses. DNA was extracted from samples obtained from herbarium specimens representing 26 taxa, including 22 accessions of Coniogramme, 2 accessions of Cryptogramma, one accession of Syngramma, and one accession of Taenitis (Table 2). Herbarium material was obtained in Herbaria P, PE, and ALTB. Furthermore, 18 accessions of 14 Coniogramme taxa, 16 accessions of 10 Cryptogramma taxa, 1 accession of Llavea, 1 accession Actiniopteris, and 1 accession Onychium were downloaded from Genbank (NCBI, National Center for Biotechnology Information, https:// www.ncbi.nlm.nih.gov/) (Table 2). Accessions of Syngramma, Taenitis, Actiniopteris, and Onychium were used as the outgroup.
DNA isolation was performed using a DiamondDNA Plant kit (ABT, Russia) according to the manufacturer's protocol. RbcL plastid DNA regions were amplified using the Biomaster HS-Taq PCR-Color (2x) (Biolabmix, Russia) with primers according to the Korall et al. (2006). PCR amplification was carried out according to the following parameters: initial denaturation for 5 min at 95 °C, followed by 35 amplification cycles: 30 s at 94 °C, 30 s at 52 °C, 90 s at 72 °C; postelongation for 7 min at 72 °C. PCR products was purified using MAXLIFE MagnetDNA kit (MVM-Diagnostic, Russia). Sanger sequencing was perfomed on an automatic sequencher ABI Prism 3130xl (Applied Biosystems, U. S. A.).
All sequences were aligned using ClustalX 2.1 (Larkin et al., 2007), and the resulting alignment was then refined manually in Geneious Prime 2022.2.2 (Biomaters Ltd., U. S. A.).
Sequence evolution models were evaluated using General Time-Reversible (GTR) with gamma
distribution and invariant sites with the aid of jModelTest2 (Darriba et al., 2012). Bayesian analyses were conducted with MrBayes 3.2.6 with four Markov chains for 1 million generations, sampling trees every 250 generations with 250 000 burn-in length (Huelsenbeck, Ronquist, 2001).
Table 2
Sources of the material and GenBank accession numbers for rbcL sequences of the taxa under the study
No. Taxon rbcL GB Acc. NR / base pair Author Locality Voucher
1 Actiniopteris radiata Link AF336100/ 1325 bp Gastony, Johnson (2001) Olduvai Gorge, Tanzania Gastony 12-97102
2 Coniogramme affinis OQ984939 / 1195 pb Present study China, Beside the Eagle Ditch in Yongxing Township. Baoxing County. Sichuan Province PE00587617
3 Co. affinis OQ984938 / 1195 pb Present study China, Shaanxi Province PE00587604
4 Co. africana OQ984934 / 1195 pb Present study Cameroon, Southwest region, Mt Kupe, Kupe Village P00968223
5 Co. caudiformis OQ984942 / 1195 pb Present study China. Hunan Province. Liuyang County Dawei Mountain Forest Farm PE01769269 (type)
6 Co. emeiensis MN867753 / 1254 bp Wang et al. (2020) Badong, Hubei Yudong Wu 012
7 Co. falcipinna OQ984944 / 1195 pb Present study China, Chekiang Province PE00587754
8 Co. fraxinea MN867752 / 1233 bp Wang et al. (2020) Cangyuan, Yunnan Zhongyang Li PT-1049
9 Co. fraxinea MN867750 / 1248 bp Wang et al. (2020) Menglian, Yunnan Zhongyang Li PT-1073
10 Co. fraxinea MN867764 / 1236 bp Wang et al. (2020) Ruili, Yunnan Gangmin Zhang 1816-4
11 Co. gigantea Ching MN867760 / 1227 bp Wang et al. (2020) China, Jinping, Yunnan Province Junwen Zhao Z2018070805
12 Co. gigantea MN867751 / 1236 bp Wang et al. (2020) Maguan, Yunnan Zhongyang Li PT-1195 pb
13 Co. intermedia var. glabra OQ984947 / 1195 pb Present study [from the label] PE828500
14 Co. intermedia var. Glabra OQ984946 / 1195 pb Present study China. Tibet Autonomous Region. Tongmai, Bomi County PE00587774
15 Co. intermedia var. intermedia OQ984945 / 1195 pb Present study [from the label] PE00587667
16 Co. intermedia var. intermedia OQ984948 / 1195 pb Present study China, Hunan Province PE01769257
17 Co. japonica AB574813 / 1205 bp Ebihara et al. (2010) -
18 Co. japonica MN867756 / 1236 bp Wang et al. (2020) Hezhou, Guangxi Rihong Jiang HZ058
19 Co. japonica OQ984930 / 1195 pb Present study Japan, Izu peninsula, Shizuoka Prefecture, Kamo District, Nashimoto, river valley Kawazu, Fuji-Hakone-Izu National Park. 35° ALTB1010004712
Table 2 (continued)
No. Taxon rbcL GB Acc. NR / base pair Author Locality Voucher
20 Co. jinggangshanensis Ching et K. H. Shing OQ984931 / 1195 pb Present study China, Jiangxi Province PE01769311
21 Co. jinggangshanensis OQ984932 / 1195 pb Present study [from the label] PE00598511
22 Co. lanceolata MN867741 / 1290 bp Wang et al. (2020) Yunxian, Yunnan Caihong Wang 20181014-2-1
23 Co. lanceolata MN867763 / 1236 bp Wang et al. (2020) Longling, Yunnan Gangmin Zhang 1820-7
24 Co. lanceolata MN867742 / 1293 bp Wang et al. (2020) Yunxian, Yunnan Caihong Wang WZ2018072902-01
25 Co. longissima Ching et Kung OQ984937 / 1195 pb Present study China, Hunan Province PE01769246
26 Co. maxima Ching et K.H. Shing OQ984943 / 1195 pb Present study China, Hunan Province PE01769317
27 Co. merrillii Ching MN867758 / 1005 bp Wang et al. (2020) China, Wuzhishan, Hainan Province Wuzhishan Fern Survey 309
28 Co. merrillii MN867744 / 846 bp Wang et al. (2020) China, Lingshui, Hainan Province Shiyong Dong 544-1
29 Co. petelotii MN867761 / 1236 bp Wang et al. (2020) Jinping, Yunnan Junwen Zhao Z2018070806
30 Co. procera MN867754 / 1233 bp Wang et al. (2020) China, Nujiang, Yunnan Province Xianchun Zhang 6344
31 Co. pubescens OQ984940 / 1195 pb Present study Yunnan, Shweli-Salwin divide, Coll. George Forrest № 24628 PE01715084
32 Co. pubescens OQ984941 / 1195 pb Present study India. Simpla. Coll: Zhang Xianchun PE01715075
33 Co. robusta OQ984929 / 1195 pb Present study China. Hunan province. Tongdao Dong Autonomous County of Dupo Township PE00598372
34 Co. rosthornii MN867745 / 1194 bp Wang et al. (2020) China, Yangbi, Yunnan Province Junwen Zhao ZY17082110
35 Co. rosthornii OQ984936 / 1195 pb Present study China. Sichuan Province. Sanlanggang. Leibo County PE00598461
36 Co. rubicaulis MN867762 / 1233 bp Wang et al. (2020) China, Rongshui, Guangxi Province Caihong Wang 1825-2-3
37 Co. rubicaulis MN867755 / 1230 bp Wang et al. (2020) China, Xinyi, Guangdong Province Yuehong Yan WYD573
38 Co. sinensis Ching OQ984949 / 1195 pb Present study China, Hunan Province PE00598327
39 Co. sinensis OQ984950 / 1195 pb Present study Sichuan Province PE01363233
40 Co. suprapilosa OQ984935 / 1195 pb Present study China. Hubei Province PE01363196
41 Co. wilsonii OQ984933 / 1195 pb Present study China, Jiangxi Province PE01769248
42 Cryptogramma acrostichoides KC700093 / 1309 bp Metzgar et al. (2013) U. S. A., Alaska, Kodiak, near the transient boat harbor Studebaker 09473 (ALA)
43 Cr. acrostichoides KC700102/ 1309 bp Metzgar et al. (2013) Russia, Kamchatka, north of Kamchatka peninsula, near Karaginskij Chernyagina s. n. (ALA)
Table 2 (continued)
No. Taxon rbcL GB Acc. NR / base pair Author Locality Voucher
44 Cr. bithynica S. Jess KT000629/ 1309 bp Metzgar et al. (2016) Turkey, Uludag, silicate scree slope on NNE side of mountain Jessen SJ-3820 (ALA)
45 Cr. brunoniana KC700082 / 1309 bp Metzgar et al. (2013) China, Xizang (Tibet) Province, Baxoi Xian, Anjiu La (pass), N of Rawu (Raog) Boufford 29733 (GH)
46 Cr. brunoniana KC700081 / 1309 bp Metzgar et al. (2013) Taiwan, NanTou County, Mt. ShihMen Kuo 455 (TAIF)
47 Cr. cascadensis KC700086 / 1291 bp Metzgar et al. (2013) U. S. A., Oregon, Deschutes / Linn County boundary, McKenzie Pass Alverson s.n. (ALA)
48 Cr. cascadensis KC700087 / 1294 bp Metzgar et al. (2013) U. S. A., Washington, King County, Source Lake Lookout Trail, above Source Lake Zika 25404 (ALA)
49 Cr. crispa KT000630/ 1309 bp Metzgar et al. (2016) Russia, Dombai, North Caucasus Jessen SJ-3099 (ALA)
50 Cr. crispa KC700089 / Metzgar et al. (2013) Spain: Madrid Province, Sierra de Guadarrama, Siete Picos Pajaron s.n. (ALA)
51 Cr. fumariifolia KC700079 / 1309 bp Metzgar et al. (2013) Chile, Provincia de Nuble, Comuna de Pinto, Shangri-La Larrain 34009 (ALA, CONC)
52 Cr. gorovoii OQ984927 / 1195 pb Present study Russia, Sakhalin isl., Makarov district, north of the villages Zaozernoe, Southern slope of Mount Ostrovskaya, along the river Ugledarka, 48°24'N, 142°40'E Vaganov, Salokhin (2239-1_SSBG)
53 Cr. gorovoii OQ984928 / 1195 pb Present study Russia, Sakhalin isl., Makarov district, north of the villages Zaozernoe, Southern slope of Mount Ostrovskaya, along the river Ugledarka, 48°24'N, 142°40'E Vaganov, Salokhin (2239-2_SSBG)
54 Cr. raddeana KC700084 / 1309 bp Metzgar et al. (2013) Russia, Republic of Buryatia, Severo-Muisky range, Samokuya Naumov 1989 (NS)
55 Cr. raddeana KC700085 / 1309 bp Metzgar et al. (2013) Russia, Khabarovsky krai, 30 km north of Sofiysk Netchaev s.n. (NS)
56 Cr. sitchensis KC700103 / 1309 bp Metzgar et al. (2013) U. S. A., Alaska, between Portage and Whittier, Bering Glacier Metzgar 248 (ALA)
57 Cr. sitchensis KC700106 / 1309 bp Metzgar et al. (2013) U. S. A., Alaska, Palmer, Hatcher Pass Metzgar 249 (ALA)
58 Cr. stelleri KC700076 / 1309 bp Metzgar et al. (2013) Taiwan: NanTou County, Hohuan Shelter Kuo 492 (TAIF)
59 Cr. stelleri KC700077 / 1309 bp Metzgar et al. (2013) U. S. A., Alaska: Alexander Archipelago: Prince of Wales Island Johnson 20104 (ALA)
60 Llavea cordifolia KC700108 / 1309 bp Metzgar et al. (2013) Mexico: Hidalgo: Municipio Nicolas Flores. On road to Nicolas. Flores from Cardonal Rothfels 3025 (ALA, DUKE, MEXU)
61 Onychium japonicum (Thunb.) Kunze KU744808 / 1246 bp Zumkeller et al. (2016) Plant material was obtained from the Botanic Garden Bonn Botanic garden accession numbers «xx-0-BONN-26126»
62 Syngramma borneensis (Hook.) J. Sm. OQ98492 / 1195 pb Present study Mimika Regency, PT-Freeport Indonesia Concession Area: Tributary of the Marao river. 4° PE01578069
63 Taenitispinnata (J. Sm.) Holttum OQ984925 / 1195 pb Present study About 2 km NNE of Julatten, north Queensland. 16° PE01966567
Phylogeography
Phylogeography of taxa was reconstructed using Mesquite software (Maddison W., Maddison D., 2018). Selected characters were optimized onto the tree set obtained from the maximum likelihood
(ML) analysis of rbcL including one exemplar for each species. Jukes-Cantor distance models of DNA evolution were used with the choice of the single Linkage clustering method (with maxtrees set to 10,000). For the table with standard categorical data in Mesquite, we used three characters designating floristic kingdom, subkingdom and region. Characters were treated as unordered, categorical variables and mapped onto the phylogenetic trees obtained from the rbcL dataset to infer patterns of evolution.
3. Results
3.1. Phylogeographic analysis
The analysis of the generalized material using a global vegetation classification by R. V. Kamelin (2017) showed that the distribution of Coniogramme, Cryptogramme, and Llavea is limited to three floristic kingdoms, including nine subkingdoms and 17 regions of the Old and New World (Fig. 1). Most of the studied species (24 out of 40) are located in the largest floristic Kingdom on the Earth - the Holarctic Kingdom, nine taxa occur in both, Holarctic and Paleotropical Kingdoms, five species are distributed in Paleotropical Kingdom only, one species grow in Neotropical Kingdom and one species - in both, Holarctic and Neotropical Kingdoms.
All taxa of Coniogramme are distributed in the Old World, among them, 16 taxa are distributed in east part of Holarctis, five taxa - in both, east Holarctis and Paleotropis, and five species grow in Paleotropis. The most species occur in East Asia in Sino-Japanese Region; some of them distributed westward to Sino-Himalaian (Co. emeiensis, Co. fal-cipina, Co. intermedia var. glabra, Co. robusta (including variations), Co. rosthornii, Co. suprapilosa) and Indian (Co. affinis, Co. lanceolata) Regions and eastward to Indochinese and Malesian Regions (Co. petelotii, Co. procera, Co. pubescens), the most widespread are Co. fraxinea and Co. intermedia var. intermedia. The narrow geographic range within the single floristic region is characteristic for Co. ca-udiformis and Co. longissima (Sino-Himalaian Region), Co. japonica, Co. jinggangshanensis and Co. rubicaulis (Sino-Japanese Region) and Co. pilosa (Hawaian Region). Only two species occur
outside of East Asia and the Pacific Islands: Co. africana (African Subkingdom, Guineo-Congolian and Zambezian Regions) and Co. madagascariensis (Madagascar Subkingdom, Madagascar Region).
The genus Cryptogramma is distributed mainly in extratropical Holarctis of the Old and New World, the only species - Cr. fumariifolia is an endemic growing in Neotropis in the mountain of Andian Region with temperate climate. The most widespread is Cr. stelleri and Cr. acrostichoides, which occur in three subkingdoms of Holarctis.
The geographic range of the single species of Llavea lies in Holarctis of New World (Pacific-American Region) and Neotropis (Carribean Region).
3.2. Phylogenetic analysis of the rbcL dataset
The dataset for 40 species was comprised. The aligned data matrix for the protein-coding rbcL locus was 1195 bp in length, where "n" we used to denote empty values. The BI tree shows a similar topology with the ML tree. The posterior probability values are shown at the nodes (Fig. 2). Our results showed that all genera Coniogramme, Cryptogramma, and Llavea formed the separate clades. The assertion was confirmed that Llavea is sister to Coniogramme and Cryptogramma together. The genera Coniogramme and Cryptogramma are reciprocally monophyletic, and their representatives are united by absolute relationship (PP = 1.0).
Inside Coniogramme superclade, Co. merrillii is sister to the rest taxa and its topology is strongly supported (PP = 1.00). The rest Coniogramme taxa are grouped into two strongly supported (PP = 1.0) unequal clades, which we named Coniogramme-I-clade including 20 taxa, and Coniogramme-II-clade including 4 species. The Coniogramme-I-clade dichotomizes into the subclades of the first order (PP = 0.99) including 13 and 7 taxa respectively. The first subclade uniting 13 taxa is divided into the next two unequal subclades of the second order. First of them is divided (PP = 0.62) into two parts, one of which (PP = 0.9) remains unresolved and is characterized by the unspecific set of eight taxa, another part includes two species that are strongly supported (PP = 1.0) as monophyletic lineages -Co. affinis and Co. pubescens. The second subclade of the second order (PP = 1.0) includes three species, among them, Co. suprapilosa is sister to Co. rosthornii and Co. longissima together, and the only accession of Co. longissima nested with one of accessions of Co. rosthornii.
Fig. 1. Distribution of Coniogramme, Cryptogramme, and Llavea taxa among global floristic regions. Phylogeography of the genera is shown using Mesquite.
Fig. 2. The majority rule consensus topology resulting from the Bayesian / Markov Chain Monte Carlo analysis of the rbcL dataset. Branch length corresponds to the estimated number of substitutions. Values above branches correspond to the bootstrap values.
The second first order subclade of Coniogramme-I clade includes seven species, that are united into two moderately supported (PP = 0.61) subclades of the second order. First of them includes three well-supported lineages corresponding to three species from which Co. africana is sister to Co. lanceolata and Co. fraxinea together, and accessions of the two last species are characterized by their genetic homogeneity in rbcL gene. Another second order subclade includes two lineages: moderately supported (PP = 0.7) lineage with Co. procera and Co. petelotii and strongly supported one (PP = 1.0) with Co. gigantea and Co. rubicaulis that appear to be identical in rbcL gene.
The Coniogramme-II-clade unites four species (PP = 1.0). Two lineages correspond with two species - Co. robusta and Co. japonica, besides Co. robusta is sister to the rest species of this clade. The third unresolved lineage includes two accessions of Co. jinggangshanensis Ching et K.H. Shing and single accession of Co. wilsonii.
The Cryptogramma superclade includes two strongly supported (PP = 1.0) clades corresponded to two sections (subgenera): Cryptogramma including the most of species and Homopteris including a single species Cr. stelleri. Plastid rbcL gene has not resolved the Cryptogramma-clade, it includes four groups as equal, one of which includes accessions
of a single species - Cr. cascadensis, other groups include different species. The first group includes two lineage: strongly supported monophyletic Cr. gorovoii and moderately supported lineage including Cr. bithynica S. Jess. and Cr. crispa. In the second group, Cr. fumariifolia supported on the specific level, accessions of the Cr. raddeana formed the strongly supported monophyletic lineage within Cr. brunoniana. Finally, the third group is unresolved and includes all accessions of Cr. acrostichoides and Cr. sitchensis as equal.
3.3. Spore morphology and morphometry
Spores of all examined species of Coniogramme, Cryptogramma, and Llavea are trilete and tetrahedral or tetrahedral-globose in shape. In equatorial position, the distal part is convex to hemispherical, proximal part is flat, convex or conical. In polar position, spores are triangular, roundish-triangular or triangular-roundish in outline with straight, concave or convex sides and rounded corners. Macro-ornamentation of the spores of Coniogramme is not well defined, mostly the same on proximal and distal part of spore. Macro-ornamentation of the spores of Cryptogramma is more coarse and slightly different on proximal and distal part. Micro-ornamentation of the spore surfaces is presented by granular deposits in varying density. Three laesura arms form a clearly visible commissure without commissural flange. Laesura arms are mostly straight, long, reaching the spore corners, or short with length from to % of radius of the spore as seen in proximal-polar position. The main characters of spores of all examined taxa are shown in detail in figures 3-10 and described in the Table 3.
Based on definition of macro-ornamentation, we recognise six spore types in cryptogrammoid ferns. The first four types of ornamentation - smooth, granulate, papillate, and verrucate - are formed by perispore, exospore is smooth (types I-IV); these type are different to each other in the presence / absence and the size of the prominent elements of sculpture. The next two types of ornamentation -colliculate and tuberculate - are characterized by more coarse sculpture elements formed by thick exospore, the thin perispore conforms to exospore (types V-VI).
Type I - Proximal and distal faces smooth, without any sculpture. This type includes two narrowly endemic species of Coniogramme -Co. madagascariensis (Madagascar, Reunion) and Co. rubicaulis (Guangxi, China) (Fig. 3). Spore surface without or with sparse granular deposits. Both species are characterized by the flat proximal
and convex distal part of spore. Laesura arms are reaching spore corners, very prominent in spores of Co. madagascariensis (Fig. 3; Table 3).
Type II - Proximal and distal faces with granulate ornamentation. This type is the largest and contains 13 taxa of Coniogramme including two varieties of Co. intermedia and two varieties Co. robusta (Figs 3-6, Table 3). Spores of Co. serrulata (Blume) Fée are characterized by smooth proximal and granulate distal face (Fig. 6). Spore surface mostly with solitary or sparse granular deposits. The shape of the proximal part of spores is mostly flat or flat to convex, two taxa have spores with conical proximal part - Co. intermedia var. glabra and Co. pilosa; the distal part is hemispherical (Figs 4, 5; Table 3). Laesura arms are mostly reaching of spore corners, in Co. fraxinea they are of a half the radius of spore. Laesura arms are prominent in Co. falcipinna, Co. intermedia var. glabra, Co. pilosa, and Co. serrulata. Spores of most species of this type are depressed between laesura arms; spores of Co. fraxinea, Co. pilosa, Co. procera, Co. pubescens, and Co. robusta (both varieties) are unusual depressed what could be due to the incompletely developed wall in the spores (Figs 4-6).
Type III - Proximal and distal faces with papillate ornamentation. This type includes five Coniogramme species: Southeast Asian Co. affinis, Far-Eastern Co. japonica, and Southasian Co. rosthorni, Co. wilsonii, and Co. petelotii (Figs 6, 7). Granular deposits on spore surfaces are absent or solitary; spores of Co. petelotii covered by dense granular deposits, especially on the distal surface. The shape of the proximal part of spores is different, from flat in Co. japonica to conical in Co. affinis and Co. rosthorni; the distal part is hemispherical in all species of the type III. The laesura arms are different in length relatively spore radius in different species, very prominent in Co. affinis and Co. japonica, obscured by sculpture in Co. rosthorni (Figs 6, 7; Table 3).
Type IV - Proximal and distal faces with verrucate ornamentation. This type includes two species of two genera - Coniogramme suprapilosa and Llavea cordifolia with the spore ornamentation formed of verrucae varied in shape and size, more prominent in distal face. Spore surface without granular deposits in Co. suprapilosa and with sparse deposits in L. cordifolia. Proximal and distal parts of spores are different in shape. Spores of Co. suprapilosa are unusual depressed in proximal part between laesura arms. Laesura arms are prominent in Co. suprapilosa and quite obscured by sculpture in L. cordifolia (Fig. 8; Table 3).
Fig. 3. SEM micrographs of the spore type I: Coniogramm madagascariensis (A-C) and Co. rubicaulis (D-F), proximal and distal faces are smooth. SEM micrographs of the spore type II: Co. africana (G-I) and Co. caudiformis (J-L), proximal and distal faces are granulate. Spores in proximal view (A, C, D, G, J), spores in distal view (B, E, H, K), spores in equatorial view (F, I, L). Very prominent laesura arms (D, F). Scale bars: 20 ^m.
Fig. 4. SEM-micrographs of the spore type II: Coniogramme emeiensis (A-C), Co. falcipinna (D-F), Co. fraxinea (G-I), Co. pilosa (J-L), proximal and distal faces are granulate. Spores in proximal view (A, D, G, J), spores in distal view (B, E, H, K), spores in equatorial view (C, F, I, L). Deeply depressed areas between laesura arms (I, J, L). Scale bars: 20 цт.
Fig. 5. SEM-micrographs of the spore type II: Coniogramme intermedia var. glabra (A-C), Co. intermedia var. intermedia (D-F), Co. procera (G-I), Co. pubescens (J-L), proximal and distal faces are granulate. Spores in proximal view (A, D, G, J), spores in distal view (B, E, H, K), spores in equatorial view (C, F, I, L). Deeply depressed areas between laesura arms (F, I). Scale bars: 20 ^m.
Fig. 6. SEM-micrographs of the spore type II: Coniogramme robusta var. robusta (A-C), Co. robusta var. splendens (D-F), Co. serrulata (G-I) proximal and distal faces are granulate. SEM-micrographs of the spore type III: Co. affinis (J-L), proximal and distal faces are papillate. Spores in proximal view (A, D, G, J), spores in distal view (B, E, H, K), spores in equatorial view (C, F, I, L). Prominent thin (G) and very prominent thick (J) laesura arms. Scale bars: 20 ^m.
Fig. 7. SEM-micrographs of the spore type III: Coniogramme japonica (A-C), Co. lanceolata (D-F), Co. pe-telotii (G-I), Co. rosthorni (J-L), proximal and distal faces are papillate. Spores in proximal view (A, D, G, J), spores in distal view (B, E, H, K), spores in equatorial view (C, F, L), fragment of proximal side of spore and laesura arms (I). Dense granulate deposits on surfaces (G, H). Scale bars: 20 ^m (A-H, J-L), 5 ^m (I).
Fig. 8. SEM-micrographs of the spore type III: Coniogramme wilsonii (A-C). SEM-micrographs of the spore type IV: Co. suprapilosa (D-F), Llavea cordifolia (G-I), proximal and distal faces are verrucate. SEM-micrographs of the spore type V: Cryptogrammafumariifolia (J-L). Spores in proximal view (A, D, G, J), spores in distal view (B, E, H, K), spores in equatorial view (C, I, L), spore in equatorial-proximal view (F). The deeply depressed areas between laesura arms (F), prominent laesura arms with sculptural elements on sides (D, G), short laesura arms partly obscured by sculpture (j). Scale bars: 20 ^m (A-L).
Fig. 9. SEM-micrographs of the spore type V: Cryptogramma acrostichoides (A-C), proximal and distal faces are col-liculate Cr. brunoniana (D-F), Cr. cascadensis (G-I), Cr. crispa (J-L), proximal and distal faces are colliculate. Spores in proximal view (A, D, G, J), spores in distal view (B, E, H, K), spores in equatorial view (C, F, I), spore in equatorial-proximal view (L). Prominent laesura arms with sculptural elements on sides (A, D, J), short laesura arms partly obscured by sculpture (G). Scale bars: 20 ^m (D-F), 30 ^m (G-L), 40 ^m (A-C).
Fig. 10. SEM-micrographs of the spore type V: Cryptogramma sitchensis (A-C), Cr. gorovoii (D-F), Cr. raddeana (G-I), proximal and distal faces are colliculate. SEM-micrographs of the spore type VI: Cr. stelleri (J-L), proximal and distal faces are tuberculate, tubercle are irregular. Spores in proximal view (A, D, J), spores in distal view (B, E, H, K), spores in equatorial view (C, L), fragment of proximal side of spore and laesura arms (F, I). Prominent laesura arms with sculptural elements on sides (D, J), extremely prominent laesura arms (L). Scale bars: 10 ^m (F, I), 20 ^m (G, H, L), 30 ^m (A, D, J, K), 40 ^m (B, C, E).
Type and morphology of the spores of Coniogramme, Cryptogramma, and Llavea
Spore type Taxon Shape of spore parts in equatorial position Spore outline in polar position Ornamentation Laesura arms Granular deposits
Proximal Distal Proximal Distal Proximal face Distal face
I Coniogramme madagascariensis flat convex roundish-triangular roundish-triangular smooth smooth sinuous, prominent, reaching spore corners absent
I Co. rubicaulis flat convex triangular roundish-triangular smooth smooth straight, reaching spore corners sparse
II Co. africana flat to convex hemispherical triangular triangular granulate granulate straight, 3A of radius solitary
II Co. caudiformis flat hemispherical triangular triangular granulate granulate straight, 3A of radius solitary
II Co. emeiensis flat hemispherical triangular triangular granulate granulate straight, 3A of radius solitary
II Co. falcipinna flat hemispherical roundish-triangular triangular-roundish granulate granulate straight, prominent, reaching spore corners sparse on distal side
II Co. fraxinea convex to flat hemispherical triangular triangular granulate granulate straight, Vi of radius sparse
II Co. intermedia var. glabra conical hemispherical triangular triangular granulate granulate straight, prominent, reaching spore corners solitary
II Co. intermedia var. intermedia convex hemispherical triangular roundish-triangular granulate granulate straight, reaching spore corners sparse
II Co. pilosa conical hemispherical triangular triangular granulate granulate straight, prominent, reaching spore corners sparse
II Co. procera flat hemispherical triangular roundish-triangular granulate granulate straight, prominent, reaching spore corners sparse to abundant
II Co. pubescens convex hemispherical triangular triangular granulate granulate straight, 3A of radius absent
II Co. robusta var. robusta flat hemispherical triangular triangular granulate granulate straight, Vi of radius sparse
II Co. robusta var. splendens flat to convex hemispherical triangular roundish-triangular granulate granulate straight, reaching spore corners solitary
II Co. serrulata flat hemispherical triangular triangular smooth to granulate smooth to granulate straight, prominent, reaching spore corners sparse
III Co. affinis conical hemispherical roundish-triangular nearly roundish papillate papillate straight, prominent, reaching spore corners solitary
Table 3 (continued)
Spore type Taxon Shape of spore parts in equatorial position Spore outline in polar position Ornamentation Laesura arms Granular deposits
Proximal Distal Proximal Distal Proximal face Distal face
III Co. japónica flat hemispherical roundish-triangular triangular papillate papillate straight, prominent, reaching spore corners absent
III Co. lanceolata conical hemispherical roundish-triangular triangular-roundish papillate papillate straight, % of radius absent
III Co. petelotii convex convex to hemispherical triangular roundish-triangular papillate papillate straight, 3A of radius abundant
III Co. rosthorni conical hemispherical triangular-roundish roundish-triangular papillate papillate straight, Vi of radius, obscured by sculpture absent
III Co. wilsonii convex hemispherical triangular triangular papillate papillate straight, 3A of radius solitary
IV Co. suprapilosa convex subconical triangular triangular verrucate verrucate straight, prominent, reaching spore corners absent
IV Llavea cordifolia flat hemispherical triangular triangular verrucate verrucate obscured by sculpture sparse
V Cryptogramma acrostichoides convex hemispherical triangular triangular low- colliculate colliculate straight, prominent, reaching spore corners, with colliculae on both sides of laesura arms solitary
V Cr. brunoniana convex hemispherical triangular triangular low- colliculate colliculate straight, prominent, reaching spore corners, with colliculae on both sides of laesura arms sparse
V Cr. cascadensis convex hemispherical roundish-triangular roundish-triangular low- colliculate colliculate straight, Vi of radius, partly obscured by sculpture sparse
V Cr. crispa convex hemispherical roundish-triangular roundish-triangular low- colliculate colliculate straight, prominent, reaching spore corners, with colliculae on both sides of laesura arms sparse
V Cr. fumariifolia convex hemispherical triangular-roundish triangular-roundish low- colliculate low- colliculate straight, prominent, Vi of radius sparse
V Cr. gorovoii convex hemispherical roundish-triangular roundish-triangular colliculate colliculate straight, prominent, 3A of radius, with colliculae on both sides of laesura arms solitary
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Type V - Proximal and distal faces with colliculate ornamentation. This type is characteristic for spores of eight Cryptogramma species (Table 3). In the spores of Cr. Fumariifolia, colliculae are dense spaced, minute, almost the same size on the both proximal and distal faces (Fig. 8). In spores of Cr. acrostichoides, Cr. brunoniana, Cr. cascadensis, Cr. crispa, and Cr. sitchensis, colliculae are dense arranged, contiguous, different in size: colliculae on the iroximal face are clearly smaller of those on the distal one (Figs 8, 9). In spores of Cr. gorovoii and Cr. raddeana colliculae are densely siaced, contiguous, slightly different in size: colliculae on iroximal face are slightly smaller of those in distal one (Fig. 10). The proximal part of spores is convex, the distal part is hemispherical. Laesura arms reach siore corners in the most siecies of this tyie, two siecies have relatively short laesura arms, reaching / of radius in spores of Cr. fumariifolia and H of radius in Cr. cascadensis, the rest iarts of laesura arms are obscured by sculiture. The salient feature of Cryptogramma spores is the presence of sculptural elements on the sides of irominent laesura arms (Cr. acrostichoides, Cr. brunoniana, Cr. crispa, Cr. gorovoii) (Figs 8-10; Table 3).
Type VI - Proximal and distal faces with tuberculate ornamentation. Tubercles irregularly spaced at some distance from each other, different in size: the tubercles on the iroximal side are smaller than the tubercles on the distal one. This siore tyie has been observed only in Cryptogramma stelleri. The proximal part of spore is conical, the distal part is hemisiherical. The laesura arms are extremely prominent, up to 4.2 ^m, covered by tubercles irregular in shape and size (Fig. 10; Table 3).
Equatorial diameter (ED) was a parameter that was determined to characterize the spore size. By this parameter, spores of cryptogrammoid ferns vary from 24 to 57 ^m on average. Cryptogramma species have larger spores than Coniogramme and Llavea species: ED in Cryptogramma spores varies from 36 to 62 ^m (42-57 ^m on average), spores of Coniogramme taxa vary from 23 to 42 ^m (24-38 ^m on average), and spores of Llavea vary from 34 to 41 ^m (37.5 ^m on average) (Table 4). Among Coniogramme, the largest spores are characteristic for Co. falcipinna (mean 38 ^m), the smallest spores - for Co. rosthorni (about 24 ^m on average). Among Cryptogramma species, the largest spores are characteristic for Cr. Acrostichoides (about 57 ^m on average), Cr. cascadensis and Cr. raddeana have the smallest spores (42 ^m on average). Noteworthy is that spores of two varieties of Coniogramme
intermedia and Co. robusta are different in size. The differences in size between spores of Co. intermedia var. intermedia and Co. intermedia var. glabra (37.8 and 30.6 ^m on average respectively) are more significant than those between Co. robusta var. robusta and Co. robusta var. splendens (28.7 and 30.7 ^m on average respectively) (Table 4). The spore size does not correlate with spore types: within the same spore type, spores of different size are found.
Among the Coniogramme species, Co. falcipinna has the longest laesura arms, Co. rosthorni and Co. suprapilosa - the shortest, and Co. suprapilosa the thickest laesura arms. Among Cryptogramma species, Cr. gorovoii has the longest and thickest laesura arms, and Cr. cascadensis and Cr. raddeana -the shortest ones. Relatively long and thick laesura arms are characteristic for the Llavea cordifolia spores (Table 4).
Table 4
The main biometric characters of the spores of Coniogramme, Cryptogramma, and Llavea, Min (Mean ± SE), Max (ED - equatorial diameter, LL - length of laesura arms, LW - width of laesura arms, SE - standard error of the mean)
Spore type Taxon ED, |m LL, |m LW, |im
I Coniogramme madagascariensis 24.9(26.3 ± 0.98)28.2 14.7(15.2 ± 0.27)15.6 1.5(2.0 ± 0.12)2.3
I Co. rubicaulis 33.2(33.6 ± 0.35)34.3 14.9(17.0 ± 1.03)18.1 1.1(1.3 ± 0.11)1.5
II Co. africana 27.9(29.2 ± 0.70)30.3 14.5(15.6 ± 0.77)17.0 1.8(2.0 ± 0.12)2.2
II Co. caudiformis 29.9(32.5 ± 1.34)36.3 12.5(12.9 ± 0.37)13.6 1.1(1.4 ± 0.18)1.7
II Co. emeiensis 29.5(31.2 ± 0.44)32.6 9.1(11.1 ± 0.89)14.7 0.7(1.2 ± 0.20)1.9
II Co. falcipinna 36.2(38.0 ± 0.88)39.0 17.6(18.4 ± 0.77)19.9 1.4(1.5 ± 0.09)1.7
II Co. fraxinea 34.8(35.9 ± 0.56)36.7 12.6(13.8 ± 0.59)14.5 1.7(1.9 ± 0.12)2.1
II Co. intermedia var. glabra 27.5(30.6 ± 1.20)33.9 10.0(13.4 ± 1.15)18.2 1.3(1.8 ± 0.14)2.2
II Co. intermedia var. intermedia 32.0(37.8 ± 1.64)41.9 12.6(15.6 ± 0.80)17.9 0.9(1.5 ± 0.18)2.1
II Co. pilosa 30.2(31.9 ± 0.84)32.9 16.7(18.9 ± 1.36)21.4 1.2(1.8 ± 0.43)2.6
II Co. procera 36.8(37.0 ± 0.20)37.4 14.3(14.7 ± 0.22)15.0 0.9(1.3 ± 0.26)1.8
II Co. pubescens 34.2(34.9 ± 0.47)35.8 12.2(14.9 ± 0.48)17.3 1.4(1.5 ± 0.30)1.8
II Co. robusta var. robusta 28.1(28.7 ± 0.28)29.0 10.8(11.1 ± 0.22)11.5 1.4(1.4 ± 0.01)1.4
II Co. robusta var. splendens 29.4(30.7 ± 0.78)32.1 15.0(15.6 ± 0.27)16.3 1.4(1.8 ± 0.38)2.6
II Co. serrulata 28.8(31.6 ± 1.42)33.4 13.2(14.3 ± 0.95)16.2 1.4(1.9 ± 0.27)2.3
III Co. affinis 27.6(28.4 ± 0.49)29.2 11.9(13.8 ± 1.02)15.4 2.5(2.8 ± 0.17)3.1
III Co. japonica 31.5(34.1 ± 0.85)36.8 12.5(15.3 ± 0.70)17.4 1.7(2.2 ± 0.13)2.6
III Co. lanceolata 30.0(33.6 ± 1.15)37.0 12.4(13.4 ± 0.67)14.7 0.6(0.9 ± 0.09)1.2
III Co. petelotii 27.1(30.8 ± 0.99)34.7 14.2(15.8 ± 0.58)18.0 0.8(1.2 ± 0.10)1.6
III Co. rosthorni 23.4(23.8 ± 0.45)25.6 6.6(10.2 ± 0.80)11.9 0.8 (1.2 ± 0.08)1.4
III Co. wilsonii 27.5(30.0 ± 1.27)31.7 12.5(13.6 ± 0.82)15.2 0.8(1.1 ± 0.20)1.5
IV Co. suprapilosa 29.4(30.0 ± 0.28)30.3 9.4(10.0 ± 0.32)10.5 2.5(4.3 ± 1.20)7.1
IV Llavea cordifolia 33.8(37.5 ± 0.85)41.1 17.2(19.4 ± 0.65)21.5 1.9(2.7 ± 0.03)3.5
V Cryptogramma acrostichoides 48.9(56.8 ± 0.77)62.3 17.3(23.5 ± 0.38)26.0 1.5(1.9 ± 0.08)3.1
V Cr. brunoniana 37.9(48.4 ± 0.68)52.3 17.8(23.0 ± 0.38)25.4 1.6(2.3 ± 0.15)3.6
V Cr. cascadensis 35.8(42.6 ± 0.63)45.5 9.9(17.6 ± 0.53)19.7 0.8(1.1 ± 0.05)1.6
V Cr. crispa 42.7(47.2 ± 0.46)50.2 19.0(20.6 ± 0.27)22.6 1.5(1.7 ± 0.08)2.0
V Cr. fumariifolia 42.2(45.0 ± 1.10)49.1 16.1(18.6 ± 0.50)21.3 1.7(2.1 ± 0.23)2.5
V Cr. gorovoii 41.2(52.7 ± 1.00)58.0 18.7(25.7 ± 0.70)32.0 1.5(2.8 ± 0.11)3.4
V Cr. raddeana 40.9(43.0 ± 0.40)45.7 15.6(17.9 ± 0.35)20.0 1.3(1.6 ± 0.04)1.8
V Cr. sitchensis 48.7(52.0 ± 0.55)55.9 22.0(24.2 ± 0.26)27.4 1.1(1.9 ± 0.07)2.3
VI Cr. stelleri 40.5(47.1 ± 0.8)50.4 16.5(19.8 ± 0.7)24.0 1.4(1.8 ± 0.1)2.1
Discussion
The previous molecular phylogenetic reconstruction of the family Pteridaceae showed
the earlier differentiation of the cryptogrammoid ferns from the rest Pteridaceae: cryptogrammoid clade comprised three genera is sister to the remainder of the Pteridaceae (Schuettpelz et al.,
2007). Our results confirmed previous hypotheses of relationship between Coniogramme, Cryptogramma, and Llavea, monophyly each of them and the earlier differentiation of Llavea from the remainder cryptogrammoid: Llavea is sister to Coniogramme and Cryptogramma together, it means that Coniogramme and Cryptogramma are more closely related than each of these genera with Llavea (Zhang et al., 2005; Schuettpelz et al., 2007; Metzgar et al., 2013; Wang et al., 2020). Molecular-phylogenetic study of all three genera of the cryptogrammoid ferns was revealed (Metzgar et al., 2013).
South-East Asia is the center of species richness of the cryptogrammoid ferns: six of nine species of Cryptogramma and 24 of 40 species of Coniogramme involved in our phylogeographic analysis occur in Eastern-Asiatic Subkingdom (Sino-Japanese and Sino-Himalayan Regions) of Holarctis. From here, species can spread westward through Sino-Himalayan and Indian Regions and Southern Arabia to African continent, southward to Indomalesian Subkingdom, northward to Eastern-Siberian and Arctic provinces and further to the west through boreal regions of Eurasia and to the east to the Pacific part of the North American continent. Llavea is the earliest diverging member of cryptogrammoid, which spread to Pacific part of North American continent perhaps in Cretacious, when Laurasia was divided from the Atlantic side into the North American and Eurasian continents, and from another, Pacific side, these continents were united (Ushakov, Yasamanov, 1984). Further in isolation, Llavea cordifolia was formed as the separate species and genus in the south of the Pacific-American Region.
Coniogramme phylogeny
Our phylogenetic reconstruction based on rbcL gene confirmed that the genus Coniogramme is monophyletic, but the sections Coniogramme and Notogramme as well as Co. ser. Coniogramme and Co. ser. Serratae Ching ex K. H. Shing separated on the base of character of pinnule margins are not monophyletic.
Coniogramme merrillii
Co. merrillii was described in 1927 by E.D. Merrill as a var. coriacea Merr. belonging to Co. fra-xinea and further redescribed by Ching (1930) as a distinct species Co. merrillii. Zhang et al. (2015) in their revision of Coniogramme from Hainan province (China) detected that the type specimen of Co. merrillii is identical to South East Asian
Co. macrophylla and Co. merrillii should be treated as synonym of Co. macrophylla (Blume) Hieron. Based on plastid DNA analysis, Wang et al. (2020) established that Co. merrillii (accession from Hainan) is sister to the rest representatives of Coniogramme. They established also that both specimen of Co. merrillii from Hainan and Co. macrophylla from Java (Indonesia) have the closer number of sporangium annulus cells (17-19 and 16-18 respectively) and accepted the taxonomic treatment of Zhang et al. (2015) about synonymisation of Co. merrillii to Co. macrophylla. C. Fraser-Jenkins et al. (2015, 2017) considered Co. merrillii is the poorly developed plants of Co. fraxinea and the name Co. merrillii should be reduced as a synonym of Co. fraxinea. Our study confirmed that rbcL gene separated Co. merrillii from other members of Coniogramme and Co. merrillii should not be considered as the member of the type section and type series Coniogramme. The genetic apartness of Co. merrillii is combined with three morphological characters having diagnostic value: free veins, undulate lamina margins, and extending of hydathodes to lamina margins. But because we have not accession of Co. macrophylla from its geographic range out of Hainan, we can not confirmed the synonymisation of Co. merrillii to Co. macrophylla. And certainly we agree with Wang et al. (2020) that Co. merrillii should not be a synonym of Co. fraxinea.
Coniogramme-I clade
The large Coniogramme-I clade includes 20 taxa formally belonged to the sect. Coniogramme. The clade is divided into two unequal subclades. First of them includes 13 species and is divided into the next two subclades: the one of eight taxa (six species + two varieties of Co. intermedia), which remained unresolved (intermedia-subclade), and the second well resolved subclade of two species - Co. affinis and Co. pubescens (affinis-pubescens-subclade).
Intermedia-subclade includes taxa formally belonged to ser. Serratae of sect. Coniogramme. Plastid DNA gene rbcL has not resolved this subclade that may be due to two reasons - genetic proximity and insufficiency of using one marker rbcL for distinction of the species. According to threatment in the "Flora of China" (Zhang, Ranker, 2013), Co. caudi-formis, Co. falcipinna, ^. sinensis, ^. emeiensis are the distinct species, endemic for the different part of China, whereas Co. maxima described by Ching and Shing (1981) as the distinct species close to Co. intermedia is reduced as the synonym to Co. intermedia var. intermedia. All members of intermedia-subclade are close morphologically. The following
morphological characters are typical for intermedia- Coniogramme intermedia (including var. inter-subclade: lamina 1- or 2-pinnate, lowermost pinnae media and var. glabra) is the most widespread spe-simple pinnules or pinnate, never 2-pinnate, pinnule cies from the intermedia-subclade. Its geographic margins are serrate, pinnules abaxially are not papil- range covers the East and Southeast Asia and exlose, hydathodes extending to tooth base or slightly tends westward to Central Asia (Nepal, Pakistan, into teeth. Coniogramme emeiensis and Co. falcipin- North India) and eastward to Indonesia (Fig. 11). na are distinguished by the shape of pinnules and Distribution of the all species of this subclade does extending of hydathodes; Co. sinensis and Co. caudi- not go beyond the range of Co. intermedia. Thus, formis are distinguished by the color of the stipe the taxa of this subclade are very close to each other and rachis. All species of the intermedia-subclade both morphologically and genetically and require involved in spore analysis have spores of the II type further molecular phylogenetic study using of more with granulate ornamentation. The largest spores are of plastid and nuclear DNA loci, especially for en-characteristic for Co. falcipinna and Co. intermedia demic species. var. intermedia (ED in average is 38.0 and 37.8 ^m respectively). Two varieties of Co. intermedia have spores different in size.
Fig. 11. Distribution of Coniogramme intermedia, ^. sinensis, Co. caudiformis, Oo. emeiensis, and Co. falcipinna
Affinis-pubescens-subclade is moderately supported in rbcL phylogeny and contains strongly supported lineage corresponded with two species -Co. affinis and Co. pubescens. These species are well distinguished by dividing of lamina: 3-pinnate with 2-pinnate lowermost pinnae in Co. affinis vs 1-pin-nate with simple or pinnate lowermost pinnae in Co. pubescens (Zhang, Ranker, 2013). We indicated also the differences in ornamentation and size of spores: papillate ornamentation and small size is characteristic for Co. affinis, granulate ornamentation and larger size - for Co. pubescens. Both species occur in China, India, Myanmar, and Nepal, Co. affinis is more widely distributed in China than Co. pubescens.
Well-supported suprapilosa-subclade includes three species, from which Co. suprapilosa differs in rbcL gene from Co. rosthornii and Co. longissima. Coniogramme suprapilosa and Co. rosthornii occur in South and Central China, and Co. rosthornii is distributed wider than Co. suprapilosa. These species
are distinguished by abaxially papillose pinnules and 2-pinnate lamina in Co. rosthornii and not papillose pinnules and 3-pinnate lamina in Co. suprapilosa. Furthermore, among Coniogramme species, Co. su-prapilosa has the most different spores with verrucate ornamentation, which we considered to IV spore type, while Co. rosthornii has more simple papillate ornamentation of spores. Third species Co. longis-sima was described from Sichuan and Hubei as the separate species close to Co. maxima Ching et K. H. Shing (Shing, 1981). Zhang and Ranker (2013) considered it as synonym of Co. emeiensis. In our rbcL phylogeny, accession of Co. longissima from Hunan (China) is united with one accession of Co. rosthor-nii from Sichuan in the same lineage, the second accession of Co. rosthornii is in another lineage of the same subclade, while accession of Co. emeiensis is nested in the unresolved intermedia-subclade. According to our rbcL analysis, Co. longissima is closer to Co. rosthornii than Co. emeiensis. Thus, we refrain from synonymizing Co. longissima and Co. emeiensis
until data on other DNA regions and spore studies become available.
Another part of dichotomy of the Coniogramme-I clade includes two subclades named africana-subclade and petelotii-subclade. All these species belong to sect. Coniogramme ser. Coniogramme having entire margins of pinnules and free venation.
Africana-subclade includes Co. africana, Co. lanceolata, and Co. fraxinea, whose accessions are resolved in rbcL phylogeny as monophyletic, and Co. africana is sister to Co. lanceolata and Co. fraxinea together. Coniogramme africana is the only species from African continent presented in our phylogenetic analysis, which has been never involved in any molecular analyses. Topology of Co. africana in the phylogramm confirms its separateness from other species of these subclade. Coniogramme lanceolata and Co. fraxinea are closer to each other than to Co. africana. Coniogramme lanceolata was described as a distinct species based on the shape of pinnae and position of the hydathodes (Shing, 1981). Later, Lu (2001), Fraser-Jenkins (2008), Fraser-Jenkins et al. (2017) reduced it to the synonym of Co. fraxinea. Zhang and Ranker (2013) treated Co. lanceolata as the synonym of Co. merrillii. In 2015, Zhang et al. reduced it to Co. macrophylla together with Co. merrillii. In the phylogenetic study by Wang et al. (2020), Co. merrillii is sister to the rest species of Coniogramme, whereas Co. lanceolata is closely related to Co. fraxinea. Both Co. lanceolata and Co. fraxinea have short hairs on the abaxial side of frond, and the same number of sporangium annulus cells, indicating that these two species are closely related, but differs by the localization of hydatodes (Wang et al., 2020).
Our phylogenetic reconstruction fully confirms this conclusion and shows that Co. lanceolata and Co. fraxinea are closely related to each other, and each of them is closer to Co. africana, the only Coniogramme species occurred in African continent, than to Hainan endemic Co. merrillii or Asian Co. macrophylla. Morphology of spores does not correspond with rbcL phylogeny: spores of Co. africana and Co. fraxinea have granulate ornamentation (II type), whereas spores of Co. lanceolata are papillate (III-type), but spores of all three species are different in the laesura length relative to spore radius. Spores of all these species are clearly depressed in proximal part between laesura arms. We accepted Co. africana, Co. fraxinea, and Co. lanceolata as the distinct species.
The next subclade named petelotii-gigantea-subclade includes four species formed two linea-
ges: Co. gigantea + Co. rubicaulis and Co. procera + Co. petelotii. First of them includes four accessions which are not resolved by rbcL, the second one includes two close species.
Coniogramme gigantea and Co. rubicaulis were described by Ching (in Shing, 1981) as distinct species, from which Co. rubicaulis is related to Co. fraxinea and Co. gigantea is related to Co. rubi-caulis. Both species occur in China, Co. rubicaulis is regarded as endemic to Guangxi, Co. gigantea - as endemic to Yunnan (Shing, 1981; Zhang, Ranker, 2013). They differ from each other by colour of petioles and rachis and have the same number of sporangium annulus cells (Wang et al., 2020). We had not spores of Co. gigantea for SEM analysis and did not find any information about their ornamentation. Spores of Co. rubicaulis have no ornamentation and are smooth. Zhang and Ranker (2013) reduced Co. gigantea to a synonym of Co. fraxinea. Phyloge-netic analysis using rbcL, as well as combine analysis with using five plastid DNA markers (Wang et al., 2020) shows that Co. gigantea is closely related to Co. rubicaulis, Co. petelotii, and Co. procera but distant to Co. fraxinea.
Coniogramme petelotii was considered as a synonym of Co. fraxinea (Fraser-Jenkins, 2008; Fraser-Jenkins et al., 2015, 2017), but other taxonomists regarded it as a distinct species (Shing, 1981; Lu, 2001; Zhang, Ranker, 2013). In our rbcL-phylogram, as well as combine phylogram by Wang et al. (2020), Coniogramme petelotii and Co. procera are clustered together that indicates their close relationship, while Co. fraxinea is distant from they both. Coniogramme petelotii is similar to Co. fraxinea in the number of sporangium annulus cells (Wang et al., 2020) but slightly different from it in ornamentation of spores: granulate in Co. fraxinea (II type) and papillate in Co. petelotii (III type). Yunnan endemic Conio-gramme petelotii and more widespread Co. procera (China (Taiwan, Xizang, Yunnan), Bhutan, India, Myanmar, Nepal, Thailand, Vietnam) are different from each other by dividing of lamina, shape and spreading of hydatodes, character of pinnula margins (Zhang, Ranker, 2013) and ornamentation of spores. Morphological and phylogenetic analyses support these species as distinct but close related.
Сoniogramme-II clade
The clade united the species belonging to sect. Notogramme, which includes species having veins anastomosing to form at least some areoles on each side of midrib. In the rbcL phylogeny this clade is highly supported (PP = 1.0) and includes Co. japonica subclade and Co. robusta sister to it. Lineage of
Co. japonica including two accessions from China and Japan is moderately supported, but the topology of Co. japonica in the distinct lineage corresponds with unique for this species characters - veins anastomosing regularly to form 1 or 2 (to 3) continuous rows of areoles on each side of midrib. Plastid locus rbcL has not resolved another lineage including two accessions of ^. jinggangshanensis and one accession of ^. wilsonii (all accessions from China). This indicates that these two species are close genetically as well as morphologically: in ^. wilsonii, veins form a discontinuous row of areoles on each side of midrib and stipe is straw-colored, in ^. jinggangshanensis, veins form only 1 or 2 areoles on each side of midrib and stipe is chestnut-colored. Two species of the Coniogramme-II clade - Co. japonica and ^. wilsonii - have spores with papillate ornamentation. Topology of Co. robusta indicates its more genetic closeness with ^. jinggangshanensis, ^. wilsonii, and Co. japonica than with any of the other Conio-gramme species. Morphologically, Co. robusta belongs to sect. Coniogramme ser. Coniogramme including species with free venation of lamina. Spores of Co. robusta have granulate ornamentation instead the papillate ornamentation of spores in ^. wilsonii and Co. japonica.
Cryptogramma phylogeny
Phylogenetic reconstruction based on rbcL gene of plastid DNA showed the same result with the reconstruction based on six plastid DNA markers (Metzgar et al., 2013, 2016): the genus, as well as the sections (or subgenera) Cryptogramma and Homopteris within the genus are monophyletic. All included in our phylogenetic analysis accessions of all species are clustered into two clades related with the sections Cryptogramma and Homopteris.
Plastid gene rbcL has not resolved the section Cryptogramma, all accessions clustered in four subclades with uncertain topology. In this study we include Cr. gorovoii described from Sakhalin Island (Vaganov, Shmakov, 2007), that was not assessed in previous studies due to a lack of suitable material (Metzgar et al., 2013, 2016). In our phylogenetic studies we received the results similar to them in the studies by J. S. Metzgar et al. (2013, 2016): we recognized all taxa of the genus Cryptogramma included in the dataset as the distinct species. Surprisingly, Far Eastern Cr. gorovoi being involved in the rbcL analysis clustered with Cr. crispa (accessions from the Caucasus and Spain) and the Turkish octoploid Cr. bithynica but not with East Asian representatives of Cryptogramma. RbcL
analysis has included Cr. fumariifolia in the same clade with two East Asian species Cr. raddeana and Cr. brunnoniana, that demonstrates their relativeness and perhaps indicates the migration of an ancestor of Cr. fumariifolia from East Asia to South America in Cretaceous but not from North to South America after the Pliocene glaciation. Metzgar et al. (2013) considered Cr. fumariifolia as the earliest diverging member of the section Cryptogramma lineage. Topology of Cr. raddeana and Cr. brunnoniana accessions indicates the close relationship between these species.
Llavea phylogeny
Our study based on rbcL gene analysis confirmed the results of previous molecular phylogenetic studies (Metzgar et al., 2013, 2016; Wang et al., 2020): the single member of the genus Llavea, mainly the Central American species L. cordifolia, is the earliest diverging member of the subfamily Cryptogrammoideae sister to Coniogramme and Cryptogramma together. Coniogramme and Cryptogramma are more closely related to each other than each of these genera with Llavea.
Spores of 32 species of three genera of cryptogrammoid ferns (Coniogramme,
Cryptogramma, and Llavea) have been characterized in detail. The spores of cryptogrammoid ferns do not have the cingulum and commissural flanges characteristic of other groups of ferns of the family Pteridaceae (Palacios-Rios et al., 2017b; Vaganov et al., 2018, 2020, 2021; Chen et al., 2022). In general, the simplicity in ornamentation (smooth, granulate, and papillate) is characteristic for the spores of the most Coniogramme species, except Co. suprapilosa. More coarse ornamentation of spores differs Llavea (verrucate) and Cryptogramma (colliculate and tuberculate). Peculiarities of macro-ornamentation allows us to define six spore types: four spore types in Coniogramme (I-IV), two spore types in Cryptogramma (V and VI) and one spore type in Llavea (IV).
General morphology, ornamentation and size of spores studied here correspond with the same features previously published for these genera (Nayar, Devi, 1966, 1967; Tryon, Lugardon, 1991; Zhang G. M., Zhang X. Ch., 2003).
Nayar and Devi (1966) gave the descriptions of spores of four species using the light microscopy: Coniogramme africana, Co. fraxinea, Co. intermedia, and Co. javanica Fée, and characterized spore ornamentation of the first two species as slightly granulose. Four Coniogramme species (Co. japo-
nica, Co. macrophylla, Co. africana, and Co. pilosa) were examined and illustrated by Tryon and Lugardon (1991). According to their SEM-micrographs, spores of Co. japonica, Co. africana, and Co. pilosa have the same ornamentation as the spores of the same species studied by us. Tryon and Lugardon (1991) identified spore ornamentation of Co. japonica, Co. africana, and Co. pilosa as papillate, while we identify it for Co. japonica as papillate, for Co. africana and Co. pilosa as granulate. Our dividing of Coniogramme based on spore types does not correspond with the existing classification of this genus based on the character of pinnule margins: sect. Coniogramme with ser. Coniogramme and ser. Serratae Ching and sect. Notogramme. Ser. Coniogramme includes species having spores of three spore types: I (Co. rubicaulis), II (Co. fraxinea) and III (Co. petelotii); ser. Serratae includes the species having spores of II (Co. intermedia, Co. procera) and III (Co. rosthorni) spore types. Section Notogramme includes species with III spore type (Co. japonica). Among Coniogramme species presented in our study, Co. suprapilosa has the most different spores with verrucate ornamentation, which we considered to IV spore type. In this character, Co. suprapilosa is closer to Llavea cordifolia than to other members of the genus Coniogramme.
The SEM-micrographs of Cr. stelleri and Cr. crispa s. l. from different part of their geographic range presented by Tryon and Lugardon (1991) are very similar with ours, but they have described such ornamentation as verrucate. Nayar and Devi (1967) mentioned and illustrated spores of Cr. crispa s. l. by LM, described ornamentation as densely subverrucate-areolate with usually circular in outline verrucae, smaller around the laesura. SEM-micrographs of spores of five taxa of Cryptogramma (Cr. brunoniana, Cr. emeiensis, Cr. shensiensis Ching, Cr. brunoniana var. sinensis (Christ) G. M. Zhang, and Cr. stelleri) presented in the work of G. M. Zhang and X. Ch. Zhang (2003), show a significant similarity in ornamentation with those in our study for the same species.
According to our study, all species of the sect. Cryptogramma have spores of V type. Among them, spores of Cr. fumariifolia are most distinguished from spores of other species with this spore type by low and quite uniform colliculae on the both part of spore. Among other species, the greatest similarity in ornamentation is noted between Cr. acrostichoides, Cr. brunoniana, Cr. cascadensis, Cr. crispa, and Cr. sitchensis and between Cr. raddeana and Cr. gorovoii. Cryptogramma sitchensis, which is the
allotetraploid with progenitors Cr. acrostichoides and Cr. raddeana (Metzgar et al., 2013) has spores more similar with those of Cr. acrostichoides than with Cr. raddeana. The only member of the sect. Homopteris, Cr. stelleri, has spores that we assigned to the type VI. Thus, our grouping of Cryptogramma spores into two spore type is consistent with existing classification: all analysed species belonging to the sect. Cryptogramma have spores of V type with colliculate ornamentation, the single species of the sect. Homopteris has spores of VI type with tuberculate ornamentation.
Llavea cordifolia spores we assigned to the IV type with verrucate ornamentation. Tryon and Lugardon (1991) determined ornamentation of spores of this species as irregularly tuberculate to papillate and emphasized that the tuberculate to papillate contour is formed by perispore, not the exospore as in other spores in the Pteridaceae. According to our study of spores, Llavea is more similar to Coniogramme than to Cryptogramma, because the sculpture elements of ornamentation are formed by perispore in Llavea and Coniogramme, not by exospore as in Cryptogramma. The spores of Llavea cordifolia are especially similar in ornamentation with spores of Coniogramme suprapilosa.
Spore size based on equatorial diameter of all three genera ranges on average between 23 and 62 ^m, which corresponds to the medium-sized spores characteristic of most species of homosporous ferns (Tryon, Lugardon, 1991). We did not found in literature data on the spore size for the most species of Coniogramme and Cryptogramma. Tryon and Lugardon (1991) mentioned about range of spore size for Coniogramme 30-50 ^m (based on six species), for Cryptogramma - 43-57 ^m (based on two species), for Llavea cordifolia - 32-42 ^m. Nayar and Devi (1966) indicated range 24-30 x 40-42 ^m for Coniogramme spores (based on four species) and size 42 x 56 ^m for Cryptogramma crispa. Our range of this parameter based on measurements of spores of 21 Coniogramme species is shifted towards smaller values (25-42 ^m) in compare with Tryon and Lugardon's data and corresponds with the range indicated by Nayar and Devi (1966). Spore size of Cryptogramma based on 9 species is characterized by the larger range - 38-62 ^m in compare with Tryon and Lugardon's data. For Llavea cordifolia, we found the same range of spore size - 34-41 ^m. The very similar in ornamentation, spores of Llavea cordifolia and Coniogramme suprapilosa are significantly different in size: 33.8-41.1 ^m and 29.4-30.3 ^m respectively.
Spore size may be related to ploidy level. A positive correlation between spore size and ploidy level was earlier observed in Adiantum L. and Polysti-chum Roth (Barrington et al., 1986), Gymnocarpium Newman (Sorsa, 1980; Pryer et al., 1983), Cystopteris Bernh. (Blasdell, 1963), but other authors observed no size differences between spores of the different ploidy levels, particularly between tetraploid and diploid species (Britton, 1968). In our study, spore size does not correspond with ploidy level in the species for which it is known. The difference in spore size between two diploids Coniogramme affinis and Co. procera is greater (ED = 28 and 37 |m on average respectively) than between each of them and the hexaploid Co. pilosa (ED = 32 |m on average); tetraploids Co. africana and Co. rosthorni have spores close in size or smaller (ED = 29 and 24 |m on average respectively) than mentioned diploids and hexaploid. The spores of autotetraploid Cr. crispa have the close size (ED = 47 |m on average) with spores of diploids Cr. brunoniana, Cr. cascadensis, Cr. Raddeana, and Cr. stelleri (ED = 48, 43, 43, and 47 |m on average respectively), but they are smaller than spores of diploid Cr. acrostichoides (ED = 57 |m on average). Spores of allotetraploid Cr. sitchensis (n = 60, ED = 52 |m on average) are intermediate in size between those of the parent species Cr. acrostichoides (n = 30, ED = 57 |m on average) and Cr. raddeana (n = 30, ED = 43.0 |m on average). Two chromosome counts are indicated for Co. intermedia (n = 30 and n = 60). Perhaps, different chromosome counts belong to different varieties especially since their spores are distinguished in size: spores in var. glabra (ED = 31 |m on average) are smaller than those in var. intermedia (ED = 38 |m).
Conclusion
In this study, phylogenetic reconstruction, spore morphology, and phylogeography for most taxa of the "cryptogrammoid clade" of the large family Pteridaceae were combined. A compehensive analysis of the data on phylogeny and spore morphology revealed correspondence of general character of spore ornamentation to each genus. The Coniogramme representatives produce spores with simple ornamentation - smooth, granulate, and papillate, ornamentation of spores of Cryptogramma and Llavea is more coarse - verrucate, colliculate, and tuberculate.
Phylogenetic reconstruction based on rbsL gene of plastid DNA sequencing of 24 taxa of Coniogramme,
ten species of Cryptogramma, and one species of Llavea confirmed the previous conclusion about monophylly of each genus and close relationship between Coniogramme and Cryptogramma than between each of them and Llavea.
We established a deep divergence of Coniogramme merillii in the Coniogramme superclade: this species is the sister lineage to the remainder of Coniogramme. We revealed also the separateness of Co. suprapilosa from Co. rosthornii and Co. longissima, Co. africana from Co. lanceolata and Co. fraxinea, and Co. robusta from Co. jinggangshanensis, Co. wilsonii, and Co. japonica. We do not support synonymisation Co. longissima and Co. emeiensis, Co. lanceolata and Co. merrillii, Co. gigantea and Co. fraxinea, Co. petelotii and Co. fraxinea. We can not confirmed the synonymisation of Co. merrillii with Co. macrophylla until we have not accession of Co. macrophylla from different parts of its range. Genetic separateness of Co. suprapilosa corresponds with exceptional ornamentation of its spores. Among Cryptogramma species, the more closeness of Far Eastern Cr. gorovoi with Cr. crispa from Spain and the Caucasus and the Turkish Cr. bithynica but not with any Far Eastern species is unexpected.
In Coniogramme, the grouping of species attending the spore type does not agree with existing classification and phylogenetic hypotheses. In Cryptogramma, the grouping on the spore types corresponds with other morphological characteristics, existing classifications and molecular phylogeny.
Spore ornamentation has diagnostic value in the recognition of the cryptogrammoid taxa at the generic and section (in Cryptogramma) levels. The presented set of spore features could be used also for diagnostic of the species as the additional morphological characters, so it could be considered in the future taxonomical reviews and in the development of a classification system of the cryptogrammoid ferns, especially for Coniogramme.
East Asia (Sino-Japanese and Sino-Himalaian Regions) is the center of origin and diversity for the subfamily Cryptogrammoideae and especially for the genus Coniogramme.
Further research on the "cryptogrammoid clade" of Pteridaceae should be emphasized on detection of relativeness inside Coniogramme, especially inside the Co. intermedia-subclade including several close species.
Acknowledgements
We are grateful to curators of the Herbaria ALTB, LE, TK, and VLA for the permission of obtaining the spores from the herbarium material. We are especially grateful to Germinal Rouhan, curator of Ferns et Lycophytes of the Paris Vascular Plant Herbarium (P, Muséum National d'Histoire Naturelle, MNHN -Paris, France) for individual approach, advice on the tropical fern group and help in working with the collection, as well as curators of Herbarium of Institute of Botany of Chinese Academy of Sciences (PE,
Beijing, China) for the permission of obtaining the spores from the herbarium material.
The study was carried out within the framework of the State Assignments of the Ministry of Science and Higher Education of the Russian Federation: A. V. Vaganov, M. V. Skaptsov and A. I. Shmakov (Altai State University) - project No. FZMW-2023-0008, and within framework of the Tomsk State University Development Programm (Priority-2030): I. I. Gureyeva, A. A. Kuznetsov, R. S. Romanets (Tomsk State University) - project 2.0.12.22.
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