Calypogeia (Calypogeiaceae, Marchantiophyta) in Pacific Asia: Updates from Molecular Revision with Particular Attention to the Genus in North Indochina

Calypogeia is a genus in Pacific Asia that is difficult to classify taxonomically. These difficulties arise from (1) considering the presence of oil bodies as anatomical characters for taxonomic differentiation, (2) the wide occurrence of sibling, semicryptic and geographical vicariant taxa and (3) the inevitable need to organize new datasets for molecular genetic revision of the genus. The present study uses an integrative approach, including molecular genetic, morphological, chorological and ecological methods, to understand the taxonomy of the genus in Amphi-Pacific Asia. As a result, a set of new-to-science taxa was revealed, and the suite of morphological features necessary for reliable discrimination of the taxa was revised. These results are based on the study of a large set of ‘fresh’ collections suitable for molecular analysis and morphological comparison and include data on oil bodies. The most basal branch in Calypogeia s.l. is segregated into a new genus, Asperifolia. Descriptions of the new taxa and the key to Calypogeia in Vietnam are provided.


Introduction
Calypogeia is one of the most taxonomically complex genera in Asia, and its representatives are difficult to identify and classify [1]. These difficulties are mostly due to (1) the great taxonomic value placed on features of intracellular organelles called oil bodies for traditional (morphological) systematics (these organelles rapidly decompose, even under optimal storage conditions) and (2) the presence of a large number of semicryptic species, which differ slightly in morphology, especially in a dried state [2][3][4][5][6][7]. A review of the genus in the Sino-Himalaya based on available literature data and the study of type specimens was previously provided by our group [1]. As expected, the molecular genetic exploration undertaken in the present study revealed a number of species that need to be described as new to science, as they are not conspecific to morphologically similar taxa known from Europe and North America. In addition, the complex of taxa in the branch sister to all other Calypogeia should be considered the new genus Asperfolia. To confirm these results, we compiled a dataset of specimens for use in molecular genetic analysis. The previously obtained material from Pacific Asia that was used in the present work was only available

Results
The phylogeny of Calypogeia taxa and closely related species was inferred from two regions of chloroplast DNA, the trnG intron and trnL−F spacer and nuclear ITS2. A list of accessions from which these sequences were available is presented in Table 1. The sets of accessions for the three DNA markers overlap only partially.
For the trnG intron, the alignment of 87 sequences consisted of 685 positions, among which 217 were parsimony informative, 93 were singletons and 375 were constant sites. The base frequencies across all sites were A: 0.344, C: 0.161, G: 0.129 and T: 0.365. The maximum likelihood (ML) criterion resulted in a consensus tree with a log likelihood of −3970.540. The maximum parsimony (MP) analysis yielded two equally parsimonious trees with lengths of 573 steps, a CI = 0.557809 and an RI = 0.854570. A consensus phylogenetic tree retained under Bayesian analysis (BA), along with the MP and ML bootstrap support (BS) values and the Bayesian posterior probabilities (PP) for each node are on Figure 1 (BA 50% majority rule consensus tree is on Figure S1).
All methods resulted in an almost identical tree topology. The differences concern the relative position of some low supported branches. Calypogeia accessions are organized into 11 supported clades; however, the relationships between these clades are poorly resolved. On the TCS haplotype network of trnG sequences (Figure 2), three groups of Calypogeia taxa were resolved.    The trnL-F phylogeny is shown in Figure 4 (BA 50% majority rule tree is shown in Figure S2). The alignment of 88 trnL-F sequences consisted of 512 positions, among which 146 were parsimony informative, 77 were singletons and 289 were constant sites. The base frequencies across all sites were A: 0.354, C: 0.143, G: 0.189 and T: 0.344. The ML criterion resulted in a consensus tree with a log likelihood of −3059.604. The MP analysis yielded two equally parsimonious trees with lengths of 215 steps, a CI = 0.631579 and an RI = 0.894419. The arithmetic means of the log likelihoods from the Bayesian analysis for each sampling run were −3155.54 and −3159.26. The bars on the left (yellow) are based on within-Calypogeia distances, and those on the right are based on distances involving other genera (blue).
The ITS2 phylogenetic tree shown in Figure 5 (BA 50% majority rule tree is shown in Figure S3 Table S1 shows the p-distances for the trnG sequences as well as those for the other two markers studied between Calypogeia and species designated Asperifolia, which was previously considered to be Calypogeia. On the phylogenetic trees, the Calypogeia species clustered together and are separated by long branches from a group of other Calypogeiaceae taxa: Mnioloma and Metacalypogeia. The large gap between p-distances among the Calypogea species and the group of other species, including Asperifolia, is illustrated in Figure 3. The basal position on the phylogenetic tree is occupied by the species Mesoptychia (Jungermanniaceae), used as an outgroup, followed by Geocalyx (Geocalycaceae) and then Metacalypogeia (Calypogeiaceae).
Differences in the accessions used to produce sequences of three DNA regions do not allow the construction of a combined tree. The arrangement of Calypogeia taxa is not identical across the trees constructed from three DNA markers. Such discrepancies are due to short internode lengths, which resulted in low support values.
On the phylogenetic trees, the Asperifolia taxa are no less distant from Calypogeia than Mnioloma, another genus of Calypogeiaceae, which allows us to ascribe the rank of a genus to these liverworts.
The p-distances between Asperifolia and three other Calypogeiaceae genera involved in this study (Calypogeia, Mnioloma and Metacalypogeia), Geocalyx (Geocalycaceae) and Mesoptychia (Jungermanniaceae), as well as those between all mentioned taxa, are nearly the same ( Table 2).  The trnL-F phylogeny is shown in Figure 4 (BA 50% majority rule tree is shown in Figure S2). The alignment of 88 trnL-F sequences consisted of 512 positions, among which 146 were parsimony informative, 77 were singletons and 289 were constant sites. The base frequencies across all sites were A: 0.354, C: 0.143, G: 0.189 and T: 0.344. The ML criterion resulted in a consensus tree with a log likelihood of −3059.604. The MP analysis yielded two equally parsimonious trees with lengths of 215 steps, a CI = 0.631579 and an RI = 0.894419. The arithmetic means of the log likelihoods from the Bayesian analysis for each sampling run were −3155.54 and −3159.26. The bars on the left (yellow) are based on within-Calypogeia distances, and those on the right are based on distances involving other genera (blue).
The ITS2 phylogenetic tree shown in Figure 5 (BA 50% majority rule tree is shown in Figure S3    The basal position on the phylogenetic tree is occupied by the species Mesoptychia (Jungermanniaceae), used as an outgroup, followed by Geocalyx (Geocalycaceae) and then Metacalypogeia (Calypogeiaceae).
Differences in the accessions used to produce sequences of three DNA regions do not allow the construction of a combined tree. The arrangement of Calypogeia taxa is not identical across the trees constructed from three DNA markers. Such discrepancies are due to short internode lengths, which resulted in low support values.
On the phylogenetic trees, the Asperifolia taxa are no less distant from Calypogeia than Mnioloma, another genus of Calypogeiaceae, which allows us to ascribe the rank of a genus to these liverworts.
The p-distances between Asperifolia and three other Calypogeiaceae genera involved in this study (Calypogeia, Mnioloma and Metacalypogeia), Geocalyx (Geocalycaceae) and Mesoptychia (Jungermanniaceae), as well as those between all mentioned taxa, are nearly the same ( Table 2). Table 2. Intergeneric p-distances over sequence pairs between groups. The number of base differences per site from averaging over all sequence pairs between groups is shown.

On Calypogeia arguta Nees et Mont. and Its Relatives
The basal group of species on the phylogenetic trees separated by a long branch from other Calypogeia includes C. arguta and its relatives previously attributed to Calypogeia subg. Asperifoliae (Warnst.) R.M. Schust.
The genetic distance between this group and the rest of Calypogeia far exceeds the Calypogeia infrageneric distances (Figures 1-5 and Table S1), which allows the ranking of the Calypogeia subg. Asperifoliae into the genus Asperifolia. The distant position of Asperifolia arguta and its relatives from the bulk of Calypogeia was also shown by morphological analysis as early as 1917 by Warnstorf [8], who proposed the unranked taxon 'Asperifoliae' within Calypogeia. Later, Schuster [9], following Müller [10], proposed Calypogeia subg. Asperifoliae based on that unranked taxon. Schuster (l.c.) also showed that aside from "vague and quantitative differences" in sterile plant morphology ( [9]: 117), there is a valuable feature in the structure of the capsule wall outer layer, where the cells are of nearly the same width as inner cells and each fourth longitudinal wall is free of thickenings (versus epidermal cells nearly twice as wide as inner cells and every other longitudinal wall not being thickened). We were unable to check this feature in our Indochinese materials. Moreover, we did not genetically test the taxonomic position of Calypogeia arguta named specimens collected in East Asia; therefore, we could not identify whether 'C. arguta' from East Asia is the same taxon as that we revealed in Indochina, whether it is the same as European accessions or whether it belongs to its own species that needs to be described. Being unable to resolve this problem, we provide a description of Asperifolia indosinica in this paper. The species resembles C. arguta morphologically, although it differs in somewhat smaller cells and smaller underleaves.

On Calypogeia pseudosphagnicola Bakalin, Troizk. et Maltseva
The small-sized Calypogeia growing over Sphagnum in the raised oligotrophic Sphagnum mires (Hoohmoore) and characterized by distanced leaves was commonly automatically referred to as C. sphagnicola in floristic practice. However, in the southern half of the Russian Far East, the specimens resembling this species have larger midleaf cells than those of 'true' C. sphagnicola based on European accessions. Moreover, aside from Sphagnum mires, conditional 'C. sphagicola' in the Russian Far East was also rarely observed over moist rocky outcrops.
The molecular genetic analysis performed here showed robust differences between populations of C. sphagnicola and the species resembling it in the southern areas of the Russian Far East and was treated here as C. pseudosphagnicola, which clustered with C. sphagnicola f. paludosa (Figures 1 and 4).
The latter is genetically very distant from Calypogeia sphagnicola f. sphagnicola; therefore, f. sphagnicola and f. paludosa cannot belong to the same species. Therefore, the status of C. sphagnicola f. paludosa remains unclear, and subsequent segregation is needed.
Geographically (according to available data), the areas of the two taxa somewhat overlap. However, the vast majority of localities of C. pseudosphagnicola lie southward of the middle part of the Khabarovsk Territory (i.e., southward of the 50-th latitude), while C. sphagnicola in Pacific Asia is widely distributed in NE Asia, including the Kamchatka Peninsula and Magadan Province, although it was once found in northern Sikhote-Alin (Khab-68- [14][15][16][17][18]. It is worth mentioning that although Calypogeia sphagnicola specimens from the Russian Far East belong to the same clade as European accessions of the same species, the genetic distance between them is quite high, and the status of Far Eastern populations of the species should be revised (as they may belong to another subspecies or even species).

On Calypogeia subalpina Inoue
Although C. subalpina was described at the species rank [11], it was not recognized at that rank even by the author of the taxon, who changed it to a subspecies of C. neesiana [12]. On the trnG and trnL-F trees (Figures 1 and 4) as well as on the trnG haplotype network (Figure 2), these two species are clustered together with maximal support values; these species are absent from the ITS2 dataset. Calypogeia subalpina seems to be restricted by temperate and hemiboreal insular-peninsular Pacific Asia, whereas 'true' C. neesiana may be restricted in its distribution in Asia by the boreal and hemiboreal mainland (taiga biome). Some reports of C. neesiana from the Far East [13] may be erroneous. The typical C. neesiana has leaf cells elongated along the margin, whereas C. subalpina has longer and wider leaf margin cells in intramarginal rows. These cells sometimes appear as a rim of slightly swollen cells and resembles the rim in C. marginella Mitt., although the great variation in this parameter in C. subalpina should be noted. Another distinguishing feature of C. subalpina is common subrotund underleaves versus constantly transversely ellipsoidal underleaves in C. neesiana. The photograph from this type of specimen is provided in [1]. The specimen published in Bryophyta Selecta Exsiccata №1293 under C. neesiana subsp. subalpina (see Table 1 for the specimens examined) molecularly does not belong to the C. subalpina-neesiana complex and rather belongs to the C. tosana-yoshinagana group. Calypogeia fissa subsp. neogaea R.M. Schust. was segregated from C. fissa s. str. by Schuster to place the North American populations of the taxon [9]. Later, Bakalin [14] raised its rank to the species C. neogaea (R.M. Schust.) Bakalin and recorded it from the Kamchatka Peninsula, where the species was estimated to be confined to very special habitats: whole-year warm (due to thermal activity of neighboring volcanoes) mesotrophic mires. After that record, the taxon was recorded several times in the Russian Far East [15], with the vast majority of localities within thermal habitats. The species status of C. neogaea was accepted in [16], although one year before, in the World Liverwort Checklist [17], it was treated as a subspecies. The present molecular genetic data have shown the following: (1) C. neogaea in North America is a taxon distinct from C. fissa at the species level, (2) the populations from the Russian Far East are not the same as those of C. neogaea and (3) the new species (mostly confined to thermal habitats in the Russian Far East) should be segregated. The latter is described here under C. kamchatica, which is distanced from other congeners in Figures 1, 4 and 5 and forms a clade with two accessions of C. azurea. Moreover, Calypogeia kamchatica is not closely related to C. tosana genetically despite its similar morphology. In fact, there are five taxa that are very similar morphologically: C. fissa, C. kamchatica, C. neogaea, C. tosana (Steph.) Steph. and C. yoshinagana Steph. Calypogeia yoshinagana is discussed below, while the morphological differences between the remaining four are minor and are mostly confined to the differences in the apical part of the leaves and underleaves. C. tosana commonly has shortly bifid leaves and bisbifid underleaves, while C. fissa, C. neogaea and C. kamchatica have acute leaf apices (rarely short bidentate, other than in C. fissa, which has great variation) and commonly bifid underleaves, although commonly with a blunt tooth at one or both lateral sides (underleaves are rarely short bisbifid). The most prominent differences are in the distribution of these four taxa. Calypogeia kamchatica is confined to the Amphi-Pacific part of the Russian Far East. Although not all records are from thermal habitats, the species is the most common (if not the only one occurring) in thermal warm mires across the Russian part of the Pacific Ring of Fire (Kamchatka plus Kuril Islands) and thermal mires in Sakhalin Island. Calypogeia kamchatica is formally distributed from subarctic to boreal zones, although these definitions are 'senseless' if the species occurs in thermal mesotrophic mires where the substrate temperature is warm all year round and mires possess intrazonal characteristics. Calypogeia tosana is distributed more southward, starting from the southern part of the Russian Far East (Kunashir Island and the southern flank of the Primorsky Territory) and spreads across Korea, Japan and East China to North Indochina. The species does not occur in the mesotrophic mires (which may be due to the rarity of this habitat type southward of the 40th latitude). In contrast, it is mostly confined to habitats with disturbed vegetation cover (including roadsides and steep crumbling slopes to watercourses) and is rarely observed on decaying wood, humus and litter in steep slopes in forests. Calypogeia fissa possesses an ecology similar to C. tosana and is restricted to Europe, while (the data are somewhat incomplete and may be questionable) C. neogaea is restricted to North America.
The closest relative of Calypogeia tosana is C. yoshinagana. The latter taxon was neglected for a long time since Hattori [18] synonymized it with C. tosana. This point of view was followed in the World Liverwort Checklist [17]. Dissimilar to those latter views, C. yoshinagana was treated as a distinct species by Bakalin et al. [1] and listed in Korea by Choi et al. [19]. Calypogeia tosana and C. yoshinagana form a well-supported clade in the phylogenetic trees and are indeed quite similar morphologically, although they may be distinguished by the leaf apex that is commonly bifid in C. tosana and mostly undivided in C. yoshinagana. In addition, C. yoshinagana is characterized by (1) slightly smaller cells not exceeding 40 µm in the midleaf, although midleaf cells in C. tosana sometimes reach 50 µm wide, and (2) a higher undivided zone in the underleaves, which commonly reach two cells high, versus one cell high in well-developed C. tosana. The ecology of the two species is somewhat similar, although most accessions of C. yoshinagana are from higher elevations (cool temperate and hemiboreal belts in the mountains), while C. tosana, with a few exceptions, is mostly distributed in warm-temperate to subtropical zones (and corresponding belts) and is somewhat confined to the lowlands.

A New Calypogeia Species from California
The study of the small liverwort collection provided by J. Shevock (CAS) and collected in California (U.S.A.) revealed small Calypogeia occupying a morphologically intermediate position between the pair Calypogeia yoshinagana-C. tosana and the group Asperifolia arguta-A. indosinica. The processing of the specimen for molecular analysis revealed its strong difference from both groups (Figures 1, 4 and 5). The new species named Calypogeia shevockii occupies a basal position in a clade containing most Calypogeia with low support on the trnG phylogenetic tree and a central node on the trnG haplotype network (Figure 2). On the trnL-F and ITS2 trees, its position is variable, being closest to C. kamchatica and C. azurea or C. tosana and C. fissa. The species morphologically resembles small-sized, pale-colored C. yoshinagana, although it has smaller, slightly more deeply divided underleaves, a commonly bifid leaf apex and pale coloration. These features may lead to its similarity with C. tosana and C. fissa (the specimen was originally identified by us as the latter taxon), as those species differ in their smaller size and dull (not translucent) plants. The range of morphological variation in the species is certainly poorly understood since only one specimen is known. Other morphologically similar species are Asperifolia indosinica and A. arguta, from which the new species differs in its smooth leaf cuticle, outer cells in the stem cross section similar to those inward (versus outer cells distinctly larger in Asperifolia) and smaller leaf cells. Unfortunately, the inconsistent results obtained for different genes do not permit us to describe the relationships of C. shevockii more definitely.

On Calypogeia japonica Steph.
On the trnG tree, C. japonica is included in a basal clade containing six other species but separated from them with maximum support by a long branch ( Figure 1). This species occupied a basal position to all other Calypogeia on the trnL-F tree ( Figure 4). Calypogeia japonica is very distinct from other Calypogeia due to biconcentric oil bodies that are not known in other taxa of the genus. In the absence of oil bodies, the species may be mistaken for a morphologically similar species, C. muelleriana, due to its rounded to obtuse leaf apices and distinctly divided underleaves (although far less deeply as in C. tosana and its morphological relatives). Calypogeia muelleriana is widely distributed in the North Holarctic (one of the most common taxa in North Europe), although it is quite rare in NE Asia and does not appear to occur southward of the 50th latitude in hemiboreal and temperate Pacific Asia. In contrast, C. japonica occurs southward of the 45-47th latitudes in the insular part (South Kurils plus Japan) and from the Korean Peninsula in the continental mainland to North Indochina. However, presumably, we estimate that the area of C. japonica spreads northwards by the islands in the North Pacific until North Kurils and even probably to the Commander Archipelago, where the study of fresh material is strongly required to properly identify the specimens. Other features are minor and qualitative: C. japonica has more or less translucent shoots and wavy leaves in comparison to the opaque and more rigid shoots of C. muelleriana.

On Calypogeia pseudocuspidata Bakalin, Frank Müll. et Troizk.
This species occupies a sister position to the abovementioned clade with or without the C. subalpina-C. neesiana complex on trnG and trnL-F trees, although it has quite a different position among Calypogeia vietnamica and C. granulata on the ITS2 tree (Figures 1, 4

and 5).
Calypogeia pseudocuspidata is characterized by distinct morphology. It somewhat resembles C. cuspidata with its relatively narrow, shortly decurrent underleaves, acute to shortly bicuspidate leaf apices (although never rounded, as sometimes occurs in C. cuspidata) and the upper third of leaves turned to the ventral side of the shoot. The two species are clearly different due to larger, somewhat swollen cells along leaf (also in the underleaf, although to a lesser extent) margins, a rigid texture and a brownish color in the fresh state. Swollen cells along the leaf margin are a rare feature in the Calypogeia genus but are well pronounced in C. pseudocuspidata and C. marginella. To a lesser extent, the larger cells along the leaf margin can be observed in a suite of taxa, including C. subalpina and C. pseudointegristipula. The distribution of the taxon may be underestimated since, aside from Vietnam, it was found in the Calypogeia collection from Myanmar and may be distributed in other areas of the Sino-Himalayas.

On the Distribution of Calypogeia integristipula Steph. in the Pacific
The most unexpected record was that of the specimens named Calypogeia integristipula from the southern part of the Russian Far East, which actually belong to a previously undescribed species referred to here as C. pseudointegristipula. Therefore, the identity of specimens named C. integristipula from Pacific Asia should be questioned. Indeed, C. integristipula was described from Europe and Japan based on several specimens as indefinite syntypes [20]. Later, the taxon was lectotypified by a specimen from Germany [21], and the lectotypifacation was followed by Grolle [22]. Indeed, there are some specimens from the Russian Far East that are genetically similar to the accessions from Europe. However, these specimens arise from the continental mainland of the Far East. Two C. pseudointegristipula specimens from Sakhalin and South Kurils on trnG and trnL-F trees were placed in the same clade as "true" C. integristipula. On the ITS2 tree, the position of these species is unclear.
Morphologically, C. pseudointegristipula is similar to C. integristipula but may be distinguished by its thickened cell walls in the marginal cell rows in the leaf, distinct trigones in the leaf cells and distinctly emarginate to shortly divided underleaves. Since all specimens collected from South Kurils and the southern part of the Sakhalin Island belong to C. pseudointegristipula, we may expect its occurrence in Japan and Korea, where this species presumably may substitute for C. integristipula (the latter then remains a circumboreal taxon that does not spread to the East Asian floristic region).

On Blue Oil-Bodied Calypogeia
Deep blue oil bodies are known in several species of Calypogeia [2,7] and are easily observable when the material is fresh. The blue color of oil bodies is evident even in the field due to distinctly bluish coloration in the shoot apices. The coloration of oil bodies is due to azulene derivate production [23] and might suggest the evolutionary value of this feature and that all blue oil-bodied Calypogeia tentatively form a peculiar monophyletic group within the genus. However, it was found that blue oil-bodied Calypogeia are scattered across all phylogenetic trees; therefore, this feature might appear several times, or in contrast, the production of azulene might be suppressed in several species. Indeed, the blue oil bodies are not only found in Asperifolia and the C. pseudocuspidata-C. and integristipula-C. melleriana group and their closest relatives. To visualize their distribution across the phylogenetic tree, Calypogeia species possessing blue oil bodies are marked with asterisks in the trnG phylogenetic tree (Figure 1).

On the Genus Calypogeia in Vietnam
The last list of liverworts in Vietnam [24] contains information on eight species of Calypogeia recorded in the country in the literature (including our own papers [7] and [2,25] The reports of Calypogeia arguta, as shown in the present account, should be transferred to Asperifolia indosinica. True A. arguta appears to be restricted to Europe and does not occur in Asia. The first report of C. azurea in Vietnam was provided by Shu et al. [26]. The authors wrote that "Calypogeia azurea is immediately separated from the known species of Calypogeia in Vietnam by the blue oil bodies and bilobed underleaves." (l.c.: 414). Since that publication, the species was found not to occur in Amphi-Pacific Asia, while several other species with blue oil bodies are distributed in East Asia [7]. True C. azurea seems to be restricted to Europe (probably slightly entering Siberia by dark coniferous forests). Currently, there are three species with blue oil bodies in the Vietnam flora, while the report of C. azurea in Shu et al. [26] presumably belongs to C. sinensis.
Three more species should be added to the list of Calypogeia known in Vietnam: C. apiculata, C. pseudocuspidata and C. cuspidata. The description of C. pseudocuspidata is provided in the present paper. The morphology of C. apiculata is described and illustrated in Bakalin et al. [1]. The plants we named C. apiculata in North Vietnam may not be identical to the 'true' C. apiculata described from Java Island. We were not able to test the populations from Java genetically. Moreover, the populations from Vietnam and China (the Chinese accession citations are in Table 1) may be identical to plants with the invalid name 'C. gollanii' Steph. ex Bonner, as discussed in [1]. The occurrence of C. cuspidata is even more problematic in Sino-Himalaya than that of C. apiculata. This taxon was described by Hawaii [27] and listed under Kantius cuspidatus Steph. It is questionable whether this species may have occurred in Sino-Himalaya. Moreover, there are two more names (C. confertifolia Steph. and C. hawaica Steph.) with similar descriptions from Hawaii that may be conspecific with C. cuspidata (for further discussion, see [1]). Calypogeia cuspidata is very similar morphologically to C. apiculata, which differs in its smooth cuticle (versus finely verruculose). Other differences are minor and unstable and include no or barely (versus up to 1/3 of stem width) decurrent underleaves, more densely and subimbricate (versus contiguous to somewhat distant) inserted leaves and wider (1.4 as wide as a stem versus 1.1-1.2 as wide as a stem) underleaves.
Noticeably, all reported Calypogeia taxa were found in the northern part of the country (the genus itself was first reported in Vietnam in 2017 by Shu et al. [26], based on two species records). Calypogeia likely occurs in the southern part of the country, but there are still no data to support this hypothesis.
Therefore, ten species are now known in Vietnam, and all belong to East Asian or SE Asian floral elements. Earlier [1], we provided a key to identifying all species of Calypogeia that were known and expected in Sino-Himalaya and the surrounding areas (including North Vietnam). That key certainly did not include taxa described in the present work. Moreover, it is quite complicated to use keys containing many species, with many couplets and many morphologically similar geographic vicariants. Thus, we provide an updated key to Calypogeia and Asperifolia species known in Vietnam.
3.11.1. Key to Calypogeia and Asperifolia taxa in Vietnam 1. Underleaves as large as leaves or larger, transversely ellipsoidal, with an apex entire to shallowly emarginate, oil bodies deep blue to purple and purple brown . . . C. aeruginosa.
6. Cells along the leaf margin as large as cells inward, not swollen, with merely thin walls, plants pale greenish to greenish yellowish and bluish when fresh . . . 7

Annotated List of Calypogeia and Asperifolia taxa in Vietnam
Below, we provide a list of Calypogeia and Asperifolia species known in Vietnam that have specimens examined and corresponding references to recent descriptions and illustrations. The specimens examined are limited to one for each locality where the species was recorded. The newly described species are listed but not annotated. Descriptions in [28].

Asperifolia indosinica
Illustrations in [28] (Figures 1 and 2), [1] (Figure 2A); the present paper, Figure 6. Descriptions in [28]. Illustrations in [28] (Figures 1 and 2), [1] (Figure 2A); the present paper, Figure 6. Ecology: Open to partly shaded moist cliffs, including those near streams in oro-subtropical forests with some warm-temperate floral elements with rocky outcrops.  Ecology: Open to partly shaded moist cliffs and boulders, including those near streams in oro-subtropical forests, rarely scattered Abies delavayi stands with Rhododendron trees and Sinobambusa thickets in the understorey.   Ecology: Open to partly shaded mesic to moist cliffs and their crevices, fine soil along streams and trails, living and decaying tree trunks in oro-subtropical forests, commonly with a dense bamboo understorey. Descriptions in [1,31]. Illustrations in [30] (Figures 1-10), [31] (Figures 1 and 2) and [1] (Figures 6R-Z, AA- Ecology: Open to partly shaded moist boulders and cliffs (also covered with fine soil), including those near streams, moist trunks of living and decaying tree trunks, rarely clayish soil on steep slopes, in oro-subtropical and, rarely, tropical forests.  Ecology: Open to partly shaded moist boulders and cliffs (also covered with fine soil), including those near streams, moist trunks of living and decaying tree trunks, rarely clayish soil on steep slopes, in oro-subtropical and, rarely, tropical forests. Descriptions in [1]. Illustrations in [32] ( Figure 18) and [1] (Figures 4AD-AH and 8F-I); the present paper, Figure 11. Ecology: Open moist cliffs near watercourses to (more frequently) clayish soil in steep slopes, stream banks and trails in oro-subtropical forests. Descriptions in [2]. Illustrations in [1] (Figures 3 and 4).  Descriptions in [2]. Illustrations in [1] (Figures 3 and 4). Ecology: Open to partly shaded moist cliffs, including those near streams, moist humus on steep slopes, moist trunk bases in part shade in oro-subtropical forests and dense communities composed of Sinobambusa and Rhododendron.

On the Genus Calypogeia in Myanmar
The liverwort flora of Myanmar is poorly known. To date, no member of Calypogeia has been reported from Myanmar. In

Taxonomic Treatment
The treatment includes two groups of taxa: (1) taxa newly described in the present paper and (2) taxa whose classification should be clarified in light of newly obtained data.
Asperifolia Description. Plants are soft, greenish to whitish greenish, greatly varying in size, in loose patches, creeping, loosely attached to the substratum and 10-20 mm long and 0.8-0.4 mm wide. Rhizoids virtually absent or very few. Stem cross section transversely ellipsoidal, 100-120 µm high and 150-170 µm wide, outer cells larger than inner, 22-35 µm in diameter, in the dorsal side with thicker walls, inner cells 20-30 µm in diameter, with slightly thickened walls, trigones small concave. Leaves subhorizontally inserted, contiguous to somewhat distant, nearly plane, not decurrent dorsally, shortly, but clearly decurrent ventrally, when flattened in the slide widely ovate, well-developed 500-600 µm long and 400-700 µm wide (larger leaves commonly wider than longer), bifid, divided by U-shaped sinus into two prominently acute lobes, terminating by 1-3 superposed cells. Underleaves obliquely spreading, 1.1-1.5 times wider than the stem (smaller may be narrower than the stem) when looking in alive material, barely decurrent, deeply bisbifid (smaller may be simply bifid), undivided zone 1-2 cells high, rhizogenous area not developed or as  Description. Plants are prostrate, green when fresh and brown in the herbarium 2.5 mm wide and 10-30 mm long, rigid, loosely attached to the substratum, forming patches. Rhizoids nearly absent or very rare, colorless to brownish. Stem cross s transversely ellipsoidal, 140-250 μm high and 200-350 μm wide, outer walls thin, walls thinner than external, inward cell walls become thicker, in outer layer 25-30 diameter, inward cells larger, to 50 μm in diameter, with small, sometimes ind trigones. Leaves contiguous to subimbricate (overlapping to 1/3 of above situated obliquely inserted, convex, with apex turned ventrally, when flattened in the slide w ovate to triangular ovate, well-developed 900-1300 μm long and 700-1200 μm (larger leaves are wider than longer), not decurrent dorsally and not or slightly ven apex acute to acuminate and rarely short bicuspidate (then divided by small, U-s Description. Plants are prostrate, green when fresh and brown in the herbarium, 1.4-2.5 mm wide and 10-30 mm long, rigid, loosely attached to the substratum, forming loose patches. Rhizoids nearly absent or very rare, colorless to brownish. Stem cross section transversely ellipsoidal, 140-250 µm high and 200-350 µm wide, outer walls thin, radial walls thinner than external, inward cell walls become thicker, in outer layer 25-30 µm in diameter, inward cells larger, to 50 µm in diameter, with small, sometimes indistinct trigones. Leaves contiguous to subimbricate (overlapping to 1/3 of above situated leaf), obliquely inserted, convex, with apex turned ventrally, when flattened in the slide widely ovate to triangular ovate, well-developed 900-1300 µm long and 700-1200 µm wide (larger leaves are wider than longer), not decurrent dorsally and not or slightly ventrally, apex acute to acuminate and rarely short bicuspidate (then divided by small, U-shaped sinus). Underleaves obliquely spreading, 1.1-1.3 of stem width, shortly decurrent, divided by widely V-to U-shaped sinus into two lobes with or without additional teeth (in the vast majority of cases additional teeth very obscure or absent), undivided zone 2-4 cells high, rhizogenous area not developed or as 1-2 rows of small cells. Cells in the midleaf subisodiametric, 32-74 × 24-40 µm, thin-walled, with small but distinct concave trigones, cuticle smooth; cells along the margin are much larger than inward, 45-79 µm long and 25-41 µm wide, with thickened cell walls and noticeable thick external wall and cuticle is smooth throughout.
Plants 2022, 11, x FOR PEER REVIEW 35 of 5 sinus). Underleaves obliquely spreading, 1.1-1.3 of stem width, shortly decurrent, divided by widely V-to U-shaped sinus into two lobes with or without additional teeth (in th vast majority of cases additional teeth very obscure or absent), undivided zone 2-4 cell high, rhizogenous area not developed or as 1-2 rows of small cells. Cells in the midlea subisodiametric, 32-74 × 24-40 μm, thin-walled, with small but distinct concave trigones cuticle smooth; cells along the margin are much larger than inward, 45-79 μm long and 25-41 μm wide, with thickened cell walls and noticeable thick external wall and cuticle i smooth throughout.      Description. Plants are somewhat glistening, translucent, yellowish greenish to whitish yellowish, curly when dry, very soft, in loose mats, loosely attached to the substratum and 1.5-2.2 mm wide and 15-30 mm long. Rhizoids a few to nearly absent, if present, commonly appressed to the underleaves or obliquely spreading, separated. Stem cross section transversely ellipsoidal, well-developed ca 230 × 300 µm, outer cells distinctly smaller than inner, varying from 17 µm in diameter in the ventral side to 25 µm in diameter in dorsal side, with small to vestigial trigones, outer wall thin to thickened (in the ventral side), inner cells 30-40(-50) µm in diameter, with vestigial trigones and thin-walled. Leaves obliquely to very obliquely inserted, nearly plane to slightly convex and with leaf apex slightly turned to the ventral side, nearly contiguous, not decurrent dorsally and ventrally, dorsally insertion line sometimes arcuate, leaves in the slide widely ovate, 800-1100 × 600-1100 µm (small leaves are commonly longer than wider), leaf apex acute, rarely very short bifid. Underleaves distinctly decurrent for 1/2-2/3 of stem width when looking in alive material, obliquely spreading, in the slide rounded, 1.2-2.0 times wider than the stem, 260-450 × 380-700 µm, divided by U-shaped sinus into two lobes, lateral additional teeth absent or short and blunt, undivided zone 2(-4) cells high. Midleaf cells subisodiametric to oblong, 35-75 × 30-50 µm, thin-walled and trigones virtually absent; cells along leaf margin subisodiametric, 25-38 × 20-38 µm, thin-walled, trigones vestigial, but adjacent to external wall side small and concave; cuticle is smooth throughout.   Comment. The species differs from Calypogeia neogaea in a number of features, including (1) twisting appearance when plants are dry (due to very soft texture), (2) very thin-walled leaf cells (versus slightly thickened), (3) underleaves larger (to 2 times wider than the stem, versus to 1.5 times the stem width), (4) distinctly and prominently decurrent underleaves (versus not decurrent), (5) stem cross section transversely ellipsoidal in both species, although much more planar in C. neogaea (the same as in C. fissa) and (6) leaves are variable in both taxa but are more regularly ovate in C. neogaea versus obliquely ovatetriangular in C. kamchatica. Nevertheless, the variation in the species is poorly understood. The plants from several specimens collected in Bering Island, Commander Arch (not cited here because the identity was not confirmed genetically) possess variation in the height of the undivided zone between underleaf lobes that may attain 4-5 cells.
Illustrations in present paper: Figures 19-21.  (1) twisting appearance when plants are dry (due to very soft texture), (2) very thinwalled leaf cells (versus slightly thickened), (3) underleaves larger (to 2 times wider than the stem, versus to 1.5 times the stem width), (4) distinctly and prominently decurrent underleaves (versus not decurrent), (5) stem cross section transversely ellipsoidal in both species, although much more planar in C. neogaea (the same as in C. fissa) and (6) leaves are variable in both taxa but are more regularly ovate in C. neogaea versus obliquely ovatetriangular in C. kamchatica. Nevertheless, the variation in the species is poorly understood. The plants from several specimens collected in Bering Island, Commander Arch (not cited here because the identity was not confirmed genetically) possess variation in the height of the undivided zone between underleaf lobes that may attain 4-5 cells.
Illustrations in present paper: Figures 19-21.   Description (based on studied specimens). Plants are in loose mats, pale greenish greenish brownish in herbarium, somewhat glistening, prostrate, loosely attached to substratum, 1.5-3.3 mm wide and 15-30 mm long. Rhizoids a few to abundant, obliqu to erect spreading, brownish, separated or in unclear fascicles. Stem cross section tra versely ellipsoidal, in well-developed plants ca. 180 × 370 μm, cell walls thin, but in ventral side cell walls commonly thickened in two rows, outer cells the same size w inner or slightly smaller, 20-32 μm in diameter, inner cells to 38 × 48 μm, trigones ve small. Leaves obliquely inserted, not decurrent in both sides, contiguous to subimbrica nearly plane to slightly convex, sometimes with apex turned ventrally, when flattened the slide obliquely ovate, 600-1500 × 600-1300 μm, slightly longer than wider, apex obtu to rarely short bifid. Underleaves obliquely spreading, commonly decurrent for 1/4 stem width when looking in alive material, in the slide transversely ellipsoidal, 220-30 350-650 μm, divided by U-to V-shaped sinus in to two acute lobes, additional lateral te common, blunt to acute, sometimes the same length with main lobe and then underleav bisbifid. Midleaf cells are thin-walled, with small concave trigones, oblong, 50-85 × 45μm; along margin 38-80 × 30-50 μm, thin-walled, with concave small trigones, trigon adjacent to external side sometimes concave.  Description (based on studied specimens). Plants are in loose mats, pale greenish to greenish brownish in herbarium, somewhat glistening, prostrate, loosely attached to the substratum, 1.5-3.3 mm wide and 15-30 mm long. Rhizoids a few to abundant, obliquely to erect spreading, brownish, separated or in unclear fascicles. Stem cross section transversely ellipsoidal, in well-developed plants ca. 180 × 370 µm, cell walls thin, but in the ventral side cell walls commonly thickened in two rows, outer cells the same size with inner or slightly smaller, 20-32 µm in diameter, inner cells to 38 × 48 µm, trigones very small. Leaves obliquely inserted, not decurrent in both sides, contiguous to subimbricate, nearly plane to slightly convex, sometimes with apex turned ventrally, when flattened in the slide obliquely ovate, 600-1500 × 600-1300 µm, slightly longer than wider, apex obtuse to rarely short bifid. Underleaves obliquely spreading, commonly decurrent for 1/4 of stem width when looking in alive material, in the slide transversely ellipsoidal, 220-300 × 350-650 µm, divided by U-to V-shaped sinus in to two acute lobes, additional lateral teeth common, blunt to acute, sometimes the same length with main lobe and then underleaves bisbifid. Midleaf cells are thin-walled, with small concave trigones, oblong, 50-85 × 45-63 µm; along margin 38-80 × 30-50 µm, thin-walled, with concave small trigones, trigones adjacent to external side sometimes concave. Comment. The species differs from C. neogaea in its softer plant texture (versus more or less stout), leaves planar to somewhat convex (versus slightly canaliculate), leaf apex sometimes turned ventrally (versus never turned), shortly decurrent underleaves (versus not or barely decurrent), underleaves commonly bisbifid (versus never or rarely bisbifid, although commonly with blunt lateral teeth on each side). Additionally, leaf cell walls are somewhat thickened in C. neogaea but are thin in C. fissa. It is worth mentioning that the variability in this parameter should be tested along with the variability in leaf cell size (because data suggest that leaf cells are larger in C. fissa than in C. neogaea).
Illustrations in present paper: Figure 22.
Plants 2022, 11, x FOR PEER REVIEW somewhat thickened in C. neogaea but are thin in C. fissa. It is worth mentioning t variability in this parameter should be tested along with the variability in leaf c (because data suggest that leaf cells are larger in C. fissa than in C. neogaea). Illustrations in present paper: Figure 22. Description. Plants are whitish greenish, prostate to ascending, freely gemmip loosely attached to the substratum, somewhat opaque, in loose mats, 1.2-2.5 mm and 10-20 μm long and merely soft. Rhizoids abundant, obliquely to erect spread unclear grayish fascicles. Stem cross section transversely ellipsoidal, well-develope ca 120 × 180 μm, outer cells with thickened walls (not so prominently to thin-walled ventral side), inner cells thin-walled to slightly unequally thickened, outer cells in th sal side the same size with inner cells, 22-30 μm in diameter, in the ventral side out 20-25 μm in diameter, trigones small to vestigial throughout. Leaves obliquely in dorsally insertion line somewhat arcuate, vernally leaves distinctly decurrent for of stem width, plane to canaliculate or concave, when flattened in the slide ob Calypogeia shevockii Bakalin et Troizk., sp. nov. Description. Plants are whitish greenish, prostate to ascending, freely gemmiparous, loosely attached to the substratum, somewhat opaque, in loose mats, 1.2-2.5 mm wide and 10-20 µm long and merely soft. Rhizoids abundant, obliquely to erect spreading in unclear grayish fascicles. Stem cross section transversely ellipsoidal, well-developed stem ca 120 × 180 µm, outer cells with thickened walls (not so prominently to thin-walled in the ventral side), inner cells thin-walled to slightly unequally thickened, outer cells in the dorsal side the same size with inner cells, 22-30 µm in diameter, in the ventral side outer cells 20-25 µm in diameter, trigones small to vestigial throughout. Leaves obliquely inserted, dorsally insertion line somewhat arcuate, vernally leaves distinctly decurrent for 1/2-2/3 of stem width, plane to canaliculate or concave, when flattened in the slide obliquely ovate, 800-1000 × 700-1000 µm, mostly as long as wide, apex commonly acute, although in 20-25% leaves bifid, divided by shortly U-shaped sinus. Underleaves obliquely spreading, not or barely decurrent, 1.2-1.6 as wide as the stem when looking in alive material, in the slide 200-350 × 320-450 µm, mostly bisbifid or with prominent, although sometimes blunt lateral teeth, divided by widely U-shaped sinus, undivided zone 1-2 cells high, rhizogenous area of the same height, with 2-4 rows of small cells. Midleaf cells subisodiametric to shortly oblong, with slightly thickened walls, 30-50 × 30-50 µm, trigones vestigial, concave; cells along the margin with thin to thickened walls, subisodiametric to oblong, 35- Comment. Morphologically, this species resembles small-sized, pale-colored Calypogeia yoshinagana (which explains the original identification of the specimen as C. fissa), which differs in its smaller, more deeply divided underleaves, commonly bifid leaf apex and pale coloration. Other morphologically similar species are Asperifolia indosinica and A. arguta, from which the species differs in its smooth leaf cuticle, outer cells in stem cross section similar to those inward (versus outer cells distinctly larger) and smaller leaf cells. In trnG topology, the species occupies a basal position to all Calypogeia s.l., except Asperifolia.
Illustrations in present paper: Figure 23. Calypogeia pseudointegristipula Bakalin, Troizk. et Maltseva, sp. nov. Description. Plants are green to yellowish green in fresh condition and green brownish in herbarium, prostrate, closely attached to the sunstratum, 2-3 mm wide and 10-20 mm long, more or less rigid and opaque to somewhat glistening. Rhizoids abundant, obliquely spreading, in unclear fascicles. Stem cross section transversely ellipsoidal, in well-developed sems ca 350 × 600 µm, outer cells thin-walled in the ventral side, slightly thickened in the dorsal side and distinctly thick in the inner part, trigones small, concave, outer cells 20-30 µm in diameter, inner cells 25-40 × 25-30 µm. Leaves imbricate, obliquely inserted, dorsally insertion line arcuate, ventrally not or shortly decurrent, slightly convex or nearly plane, but with apex turned to the ventral side, when flattened on the slide widely obliquely ovate, 900-1400 × 100-1800 µm, distinctly wider than longer, apex rounded to somewhat obtuse. Underleaves appressed to the stem, decurrent for 1/3-1/2 of stem width, transversely ellipsoidal, shortly emarginate to distinctly divided by semicrescentic sinus into two obtuse lobes, undivided zone 10-12 cells high, rhizogenous area well developed, 500-800 × 1000-1300 µm. Midleaf cells subisodiametric, more or less thin-walled, trigones distinct, small to moderate in size, concave, 37-60 × 33-60 µm, cuticle finely verruculose; cells along the margin with thickened external wall and other walls slightly to distinctly thickened, subisodiametric to oblong, 35  Comment. This species is probably one of the most common taxa of the genus in the southern part of the Russian Far East (Primorsky Territory, Sakhalin Island, South Kurils) and was previously misidentified as C. integristipula. C. pseudointegristipula differs from C. integristipula in its (1) distinctly emarginate to divided underleaves and (2) distinct trigones in the leaf cells, commonly distinct thickened cell walls along the leaf margin and commonly fine verruculose cuticle. Here, we limited the list of specimens examined to those specimens used for molecular analysis, although the distribution of the taxa is likely much wider.
Illustrations in present paper: Figures 24 and 25.

Morphology Comparison
The material used in the present study came from our own field research, herbarium specimens and type specimens stored in various herbaria (the most valuable are G, NICH, TNS, and VBGI). The vast majority of Calypogeia specimens that were collected within the last 10 years were studied under 'fresh' conditions for oil body parameters; in many cases,

Morphology Comparison
The material used in the present study came from our own field research, herbarium specimens and type specimens stored in various herbaria (the most valuable are G, NICH, TNS, and VBGI). The vast majority of Calypogeia specimens that were collected within the last 10 years were studied under 'fresh' conditions for oil body parameters; in many cases, oil bodies were photographed and then described. These results have led to understandings of whether molecular genetic data correlate with the differences in oil body number, size and color. The inclusion of information on oil bodies into descriptions helps us understand if the newly revealed taxa are indeed different morphologically or are cryptic. Calypogeia, as a genus, does not have many gametophyte morphological features that could be used in a simple identification practice; moreover, many distinguishing traits are quantitative. The main attention was given to the following characteristics: (1) oil body features, where the most readily observed are color (colorless versus brownish, blue and purple) and the presence of a central 'eye' (C. japonica only); (2) characters of the leaf apex (rounded to acute and bidentate); (3) shape of underleaves (rounded to bifid and bisbifid); (4) shoot transparency when plants are alive (plants translucent to opaque); (5) rigidity of plants (rigid to soft); (6) leaf three-dimensional shape (planar, convex, concave, with apex turned to the ventral side or not, undulate along margin, etc.); (7) the undivided zone in the underleaf between the underleaf base (above microcellous zone providing rhizoids) and the sinus (a middle sinus of three, if underleaves are bisbifid); (8) comparative size of outer and inner cells in the stem cross section (larger that inward or nearly the same); (9) comparative size and shape of cells along the leaf margin (larger than inside, swollen, elongated along margin, with thickened cell walls); (10) midleaf cell size; (11) leaf surface feature (smooth, versus papilllose or verruculose cuticle); and (12) the presence of trigones in the midleaf (actually they are always present, but sometimes are very small) and the thickness of leaf cell walls.
All studied specimens had preliminary names prior to molecular phylogenetic studies. In addition, we had a series of specimens with apparently the same morphological parameters, and some of them were selected for molecular genetic comparison. Since the taxonomic position of specimens was sometimes altered when the molecular analysis was performed, the names were corrected. For instance, all accessions of C. kamchatica were originally named C. neogaea, and when the necessity to describe the new taxon became obvious, the names were changed.

DNA Isolation, Amplification, and Sequencing
DNA was extracted from dried liverwort tissue using the NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Nordrhein-Westfalen, Germany). Amplification of chloroplast trnG-intron, chloroplast trnL-F spacer and nuclear ITS2 was performed using an Encyclo Plus PCR Kit (Evrogen, Moscow, Russia) with the primer pairs (forward and reverse) listed in Table 3. The isolated DNA was dissolved in TE buffer and stored at −20 • C. DNA concentration was measured with a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA).
The polymerase chain reaction was performed in a total volume of 20 µL, including 1 µL template DNA, 0.4 µL Encyclo polymerase, 5 µL Encyclo buffer, 0.4 µL dNTP-mixture (included in Encyclo Plus PCR Kit), 13.4 µL (for trnG, trnL-F)/12.4 µL (for ITS2) doubledistilled water (Evrogen, Moscow, Russia), 1 µL dimethylsulfoxide/DMSO (for ITS2) and 0.4 µL of each primer (forward and reverse, at a concentration of 5 pmol/µL). Polymerase chain reactions were carried out using the following program: 180 s initial denaturation at 95 • C, followed by 30-40 cycles of 30 s denaturation at 94 • C, 20 (trnL-F)-30 s (ITS2, trnG) annealing at 56 • C (trnG), 58 • C (trnL-F) 60 • C or 64 • C (ITS2) and 30 s elongation at 72 • C. Final elongation was carried out in one step of 5 min at 72 • C. Amplified fragments were visualized on 1% agarose TAE gels by EtBr staining and purified using the Cleanup Mini Kit (Evrogen, Moscow, Russia). The DNA was sequenced using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) with further analysis of the reaction products following the standard protocol on the ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) in the Genome Center (Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow). Table 3. Primers used in the polymerase chain reaction (PCR) and cycle sequencing reactions.

Phylogenetic Analyses
For the molecular phylogenetic study, three markers were used: nuclear 5.8S rRNA and ITS2 gene and plastid trnL-F region and trnG gene. Datasets were aligned by MAFFT using the E strategy [39] and then adjusted manually in BioEdit ver. 7.2.5 [40]. The absent parts of the sequences were coded as missing.
MP analysis for all datasets included 1000 bootstrap replicates, default settings were used for all other parameters, and gaps were treated as partial deletions with a site coverage cut-off of 95%.
For the ML analysis with 1000 ultrafast bootstrap replicates [42], the best fitting evolutionary models chosen according to Bayesian Information Criterion (BIC) by IQ-TREE were K3Pu + F + G4 for trnG, TPM2 + F + G4 for trnL-F and TNe+ G4 for ITS2 datasets.
Bayesian analyses were performed by running two parallel analyses using the GTR + I + G model. For all datasets, the analysis consisted of four Markov chains. Chains were run for five million generations, and trees were sampled every 500th generation. The first 2500 trees in each run were discarded as burn-in; thereafter, 15,000 trees were sampled from both runs. Bayesian posterior probabilities were calculated from the trees sampled after burn-in. The average standard deviation of split frequencies between two runs was 0.0058 for trnG-intron, 0.0090 for trnL-F and 0.0076 for ITS2.
A haplotype network was constructed by the TCS network inference method [44] using the PopART package (http://popart.otago.ac.nz/) [45]. The PopART program automatically removes from the consideration positions having at least one N or a gap.
The infrageneric and infraspecific variability of the trnG intron, trnL-F and ITS2 were quantified as the average pairwise p-distances calculated in Mega X using the pairwise deletion option for counting gaps. All ambiguous positions were removed for each sequence pair.
The distribution of pairwise p-distances between sequences was tabulated by the Assemble Species by Automatic Partitioning (ASAP) program (https://bioinfo.mnhn.fr/ abi/public/asap/asapweb.html, last accessed 15 February 2022) [46] with default settings and a p-distance model.