­Evolution of the most species-rich family of simple thalloid liverworts (Aneuraceae): a time-calibrated perspective into its evolutionary history and diversification

doi:10.21203/rs.3.rs-2514207/v1

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Evolution of the most species-rich family of simple thalloid liverworts (Aneuraceae): a time-calibrated perspective into its evolutionary history and diversification

https://doi.org/10.21203/rs.3.rs-2514207/v1

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Aneuraceae is the largest family of the simple thalloid liverworts. Despite simple morphological features, this family is well-known for its unique ecology, cryptic diversity, and species-rich lineages. In this study, we explored the evolutionary history and diversification of the Aneuraceae. Investigating the effects of fossil calibrations on divergence time estimates and how recognizing cryptic species impacts macroevolutionary analyses. We used publicly available nucleotide sequences to reconstruct chronograms of Aneuraceae with representatives from subclass Metzgeriidae as outgroups. We also examined the consequences of different calibrations in chronogram reconstruction. Then, the resulting time-calibrated phylogeny was employed to reveal diversification dynamics with lineage through time plots (LTTPs), γ-statistics, and Bayesian Analysis of Macroevolutionary Mixtures (BAMM). The Aneuraceae had originated and created diversity since the Lower Carboniferous (352.75 Mya, 95%HPD: 505.79–264.59) to Upper Devonian. Lineage diversification of Aneuraceae species was consistent with late bursts of speciation with two significant rate shifts in genus Riccardia: one at the crown node of the genus and the other at the Neotropical clade. Including cryptic taxa resulted in an additional rate shift at the crown node of Aneura. Aneuraceae originated during the middle Palaeozoic Era, similar to other early terrestrial plants, although different fossil calibrations significantly affected the estimation of divergence dating. Two speciation rate shifts in Riccardia could be the results of asexual reproductive structure in this genus and climate changes in the Neotropics. Ecological conditions at the origin of the family and association with fungi might be associated with the increased diversification in this family.

Aneuraceae

BAMM

Chronogram

Diversification

Evolution

Fossil calibration

Among the thalloid liverworts, Aneuraceae H. Klinggr. Is the largest simple thalloid liverwort family, with 325 species worldwide (Söderström et al. 2016). Aneuraceae currently includes five genera (Fig. 1): Afroriccardia Reeb & Gradst., endemic to the Madagascan region; Aneura Dumort., a cosmopolitan genus; Lobatiriccardia (Mizut. & S. Hatt.) Furuki, occurring in the southern hemisphere; Riccardia Gray, another cosmopolitan genus; and Verdoornia R.M. Schust., which is endemic to New Zealand (GBIF 2022). Most members of Aneuraceae form mycorrhiza-like relationship inside a thallus, except for species in the genus Riccardia (Duckett and Ligrone 2008; Preußing et al. 2010a). The family also includes a completely myco-heterotrophic liverwort, Aneura mirabilis (formerly placed in its own genus Cryptothallus Malmb.). Because of their minimal features and mycorrhizal-like symbiosis, these simple thalloid liverworts are often a subject of study to understand land plant evolution (Shaw et al. 2011).

Despite its role in investigating land plant evolution, the origin, and phylogenetic relationships of Aneuraceae is not fully understood. The phylogenetic relationship of this liverwort family has been studied in various aspects. First, the phylogenetic placement of Cryptothallus in the Aneura was proposed by Wickett and Goffinet (2008). After that, an overview and character evolution of Aneuraceae was outlined by Preußing et al. (2010b). Recently, a worldwide molecular phylogeny of Riccardia was reconstructed and revealed the new monotypic genus Afroriccardia (Rabeau et al., 2017). However, none of these studies has addressed the origin and diversification of the family as a whole.

Part of the difficulty in placing the origin of Aneuraceae comes from the paucity of fossil records. Current methods of chronogram reconstruction, such as the Fossilized Birth-Death (FBD) process (Heath et al., 2014), allow explicit inclusions of fossil records for calibration points. For subclass Metzgeriidae, of which the family Aneuracea is a part, only a few macrofossils are available: 1) Metzgeriothallus sharonae Hernick, Landing & Bartowski from the Plattekill Formation of the eastern Hamilton Group in the United States, Late Middle Devonian (Givetian, 382–388 Mya) (Hernick et al. 2008), 2) Riccardiothallus devonicus C.Q. Guo, D. Edwards, P.C. Wu, Duckett, Hueber & C.S. Li from the Posongchong Formation in the China, Early Devonian (Pragian, 407–411 Mya), and 3) Riccardiothallus palmatus P.C. Wu & C.Q. Guo from the Yixian Formation in China, Early Cretaceous (approximately 125 Mya) (Guo et al., 2012, 2017). These taxonomic placements within Aneuraceae remain controversial and their use in divergence time estimation has been discouraged (Tomescu et al. 2018; Feldberg et al. 2021). However, a molecular dating can also be helpful in fossil placements in many taxonomic groups (Wiens et al. 2010).

Phylogenetic uncertainty and the presence of cryptic species – morphologically similar species-level lineages that are not recognized under current taxonomic treatments (Struck et al. 2018) – present additional challenges in studying the evolutionary history of Aneuraceae. Wickett and Goffinet (2008) used multi-locus data to show that the distinctly myco-heterotrophic Cryptothallus mirabilis Malmb. was nested within the genus Aneura, despite their different morphology and physiology. Moreover, the type species Aneura pinguis (L.) Dumort. also appears to comprise a paraphyletic group of cryptic species, according to various studies on their biogeographic patterns (Shaw, 2001), isozyme analysis (Bączkiewicz and Buczkowska 2005), morphometric differentiation (Buczkowska et al. 2006), PCR-RFLP markers (Wachowiak et al. 2007), the composition of volatile compounds (Wawrzyniak et al. 2014), ISSR markers (Buczkowska et al. 2016), and DNA barcoding with ecological aspect (Bączkiewicz et al. 2017). Aneura was not the only genus with hidden diversity. Rabeau et al. (2017) also illustrated the phenotypic plasticity in the genus Riccardia and introduced the novel genus Afroriccardia, described based on molecular and morphological evidence. These recent studies highlight the complex evolutionary history and the need for a more thorough phylogenetic evaluation of Aneuraceae.

Given the diversity and complexity of Aneuraceae, we aimed to use the most currently available molecular data and paleontological records to unravel the evolutionary history of this family. More specifically, the main focuses of this study were (1) to estimate the timing of the origin of Aneuraceae from time-calibrated relationships within Metzgeriidae with different fossil calibrations, (2) to examine the timing of diversification and rate shifts within Metzgeriidae, and finally, (3) to investigate the effect of including the cryptic species on macroevolutionary analysis. We obtained publicly available sequence data to reconstruct a chronogram with two different fossil calibration approaches. The resulting chronogram was further examined to reveal diversification dynamics within the group.

Taxon sampling, build alignment, and model selection

We selected fifty-three taxa from the family Aneuraceae and fourteen taxa from the other two families in the subclass Metzgeriidae. Sequences representing three chloroplast loci – trnK-psbA-trnH, rps4, and trnL-trnF – were retrieved from the NCBI (https://www.ncbi.nlm.nih.gov) nucleotide database (Table S1). Sequences from nuclear markers were excluded from the current analysis because the data were unavailable for many of the studied taxa. To facilitate family-level alignments, we aligned sequences from each locus for each genus independently before combining the genus-level alignments with the other genera within the family, using the Global alignment at 70 percent similarity in Geneious Multiple Alignment tools using Geneious Prime^® 2022.0.1 (http://www.geneious.com/). We divided the resulting alignment into ten partitions based on the annotation (exon, intron, intergenic spacer in each locus, Table 1). The best nucleotide substitution model was identified for each partition separately using the ModelTest-NG (Table 1) (Darriba et al. 2020).

Table 1 Characteristics of three chloroplast loci used this study

Loci (No. of sequences)	Regions	Aligned length (bps)	AIC selected models
trnK-psbA-trnH (58)	trnK-psbA intergenic spacer	179	TPM3uf+G
	psbA exon	1,062	TIM1+G
	psbA-trnH intergenic spacer	249	TVM+I+G
	trnH exon	65	TrNef+G
rps4 (67)	trnS-rps4 intergenic spacer	215	TIM1+G
	rps4 exon	610	TVM+I+G
trnL-trnF (67)	trnL-UUA intron	420	TIM1+I+G
	trnL exon	50	TIM2ef+I+G
	trnL-trnF intergenic spacer	146	TVM+G
	trnF exon	75	K80+I+G

Fossil placements and calibrations

For the fossil placement and calibration, we chose three fossils of subclass Metzgeriidae from previous studies. First, we placed Metzgeriothallus sharonae at the crown node of Order Metzgeriales at the range of 385.3 to 391.8 Mya. Second, Riccardiothallus devonicus shared several characters with extant Aneuraceae species (e.g., flat thallus, irregular branching, lack of costae, and cell size and shape). We placed it at the crown node of Aneuraceae in the range of 407.6 to 410.8 Mya. Third, Riccardiothallus palmatus was recovered from Barremain to Aptian (113.0 to 129.4 Mya). The morphological characters of R. palmatus resembled extant species of Riccardia for having irregularly dichotomous branching with entire margins. Therefore, we assigned this fossil record to the crown node of Riccardia members. To investigate the influence of our fossil calibrations on age estimates, we set the calibration points in two different ways. The first approach used only Metzgeriothallus sharonae as the calibration point because the age of this fossil record was recognized as the most reliable in this subclass (Feldberg et al. 2021). The other approach included all three fossils for calibration.

Chronogram reconstruction

We performed Bayesian divergence time estimation in the software package BEAST v2.6.3 (Bouckaert et al. 2019), using the concatenated dataset of three plastid regions from 76 species. For both fossil calibrations approaches – the single-fossil calibration and the three-fossil calibration, divergence times were estimated with the substitution models selected from ModelTest-NG for each of the ten partitions. The Optimized Relaxed Clock (ORC) model (Douglas et al. 2021) was assigned as the clock prior to this empirical parameter at the starting rate of 5.0 × 10^-4 in every partition (Aigoin et al., 2009; Huttunen et al., 2008; Patiño et al., 2013; Pokorny et al., 2011; Shaw et al., 2010). We performed the Birth-Death (BD) and the Fossilized Birth-Death (FBD) process as the tree prior. For the BD tree prior, we calibrated a single fossil to the crown node of subclass Metzgeriidae, with the range of the origin of land plant as a constraint with the nearest time to present of Metzgeriothallus sharonae ( at 385.3 Mya) with the uniform distribution. For both of FBD tree priors, age intervals of each fossil was set to their maximum and minimum age parameters in the most recent common ancestor (MRCA) prior with the uniform distribution, together with the known taxon group priors. The age constraint of the origin of land plants at 475 Mya was also set as the maximum age of the chronogram root (i.e., originFBD.t parameter), based on the oldest known cryptospores (Wellman and Gray 2000). The results from these different dating methods allowed us to evaluate the performance of FBD tree priors against the traditional BD trees from the previous studies (Feldberg et al. 2014; Laenen et al. 2014), using an expanded concatenated dataset. Additionally, we performed the Bactrian kernels from the BEASTLabs package (Yang and Rodríguez 2013; Thawornwattana et al. 2018) as the operator schedule. Eventually, the Markov Chain Monte Carlo (MCMC) was run for 800,000,000 generations, sampling every 80,000^th tree. Analyses were conducted in the High-performance Computing facility of the Faculty of Science, Kasetsart University (SciKU HPC).

The sampled posterior trees were summarized in the software LogCombiner and TreeAnnotator v2.6.3 (Bouckaert et al. 2019) by discarding the first 25 percent as a burn-in. The resulting posterior trees were used to create a maximum clade credibility (MCC) tree with median heights. The effective sample size (ESS) of each estimated parameter was examined with Tracer v1.7.1 (Rambaut et al. 2018) to ensure that all the parameters have ESS values of 200 or greater. The resulting MCC tree was visualized using the read.beast function from the phyloch package (Heibl 2008) and the strap package (Bell and Lloyd 2015) to include the international chronostratigraphic scale in the R 4.1.2 environment (R Core Team 2019).

Diversification analyses

We discarded the extinct taxa prior to this analysis and created a multi-lineage through time (mLTT) plot using drop.tip.multiPhylo and mltt.plot commands, respectively, functions from the ape package (Paradis et al. 2004) to display individual lineage-through-time plots from the hundred posterior trees as its intervals. We tested the effect of incomplete taxon sampling by calculating the γ-statistics using the mccr function from R-package phytools (Revell 2012). To detect a rate of diversification processes, we employed the approach in Bayesian Analysis of Macroevolution Mixture (BAMM) software (http://bamm-project.org) (Rabosky et al. 2014) version 2.5.0. Priors were set for each analysis with the setBAMMpriors function from the BAMMtools package with the BAMM’s minCladeSizeForShift parameter set at 10. Four MCMC chains were run for 500,000,000 generations and sampled in every 10,000^thtree. The proportion of sampled taxa in each clade was also supplied using the “sampleProbsFilename” argument for the control files to account for incomplete taxon sampling (Table S2). The data on the sample proportion allows us to recognize the incomplete taxon sampling at the genus level, based on the most recent hornworts and liverworts checklist from Söderström et al. (2016). The inclusion of the data has been shown to reduce the effect of incomplete taxa on the estimates of diversification rates (Rabosky, 2018; Sun et al., 2020).

From previous studies, Aneura pinguis was found to contain more than one taxonomic unit (Bączkiewicz et al. 2017). Since BAMM was not designed to handle cryptic species or species complexes, we performed the diversification analysis using two different sampling proportions. First, we included each sample of ten known A. pinguis clades as ten species in the tree. Second, we included only one A. pinguis to represent the whole complex. Output speciation rates were displayed with phylorate plots to examine the diversification dynamics and rate shifts within the family. We also visualized pairwise similarity in this diversification between each tip with cohort analysis and the speciation and the extinction rates of Aneuraceae through time (Fig. S5).

Divergence date estimation of Aneuraceae

The final concatenated alignment contained 3,071 bps from 76 species with 53.3% pairwise identity, 11.0% identical sites, and 30.3% missing data. The alignment was subsequently used for chronogram reconstruction. Chronograms from the different fossil calibrations and models revealed similar topologies (Fig. 2, S1 and S2). The earliest diverging lineage led to the genus Verdoornia. Then, two major clades were reconstructed. The first clade contained Afroriccardia, Aneura, and Lobatiriccardia, and the other consisted of the single genus of Riccardia. The three methods yielded different divergence time estimates (Table 2). The estimated divergence time from the only M. sharonae calibration with FBD model put the origin of Aneuraceae in the Lower Carboniferous at 352.75 (HPD: 505.8–264.6) Mya, while the same fossil calibration on Birth-Death model showed the origin in the Upper Devonian at 365.56 (HPD: 425.80–304.43) Mya. The estimate from the three-fossil calibration put the origin of the family in the Upper Devonian 381.32 (HPD: 449.8–316.9) Mya. We decided to use the chronogram from the single-fossil calibration for further analyses because of its better ESS values and congruence with the known age of the earliest land plants (Table S3).

Table 2 Divergence time estimations from the BEAST chronograms with three different calibration schemes. The numbers are in million years ago with median node ages in bold and the range of highest posterior density.

Tree models	Birth-Death	Fossilized Birth-Death	Fossilized Birth-Death
Fossil combinations	only M. sharonae calibration	only M. sharonae calibration	Three-fossil calibration
Metzgeriidae^CG	535.67–459.46–388.02	610.23–436.80–385.46	754.19–510.70–409.71
Metzgeriales^CG	468.68–422.54–385.31	589.14–427.19–385.43	701.39–479.99–407.78
Aneuraceae^CG or Verdoornia^DN	425.80–365.56–304.43	505.79–352.75–264.59	449.82–381.32–316.92
Aneura/Lobatiriccardia^MRCA	256.40–201.54–150.11	285.93–196.19–130.07	359.71–234.66–158.67
Afroriccardia^DN	340.15–278.00–215.54	392.71–271.59–190.01	466.44–318.43–224.18
Aneura^CG	61.76–47.99–36.83	71.29–48.87–33.60	86.72–57.33–39.11
Lobatiriccardia^CG	59.52–39.64–23.98	65.50– 40.01–22.68	80.12–47.03–26.57
Riccardia^CG	238.10–192.23–147.17	274.32 – 189.04–134.23	339.04–223.07–155.62

Notes:^CG = crown group, ^DN = divergence node, ^MRCA = most recent common ancestor

Lineage diversification

The log scale multi-lineages through time (mLTT) plots showed a slow accumulation of lineages up until around 250–200 Mya (Upper Triassic to Lower Jurassic), when the genus Riccardia started to diversify. The pattern was consistent with the significant positive gamma statistics (γ = 1.2336; MCCR test P < 0.0001, Fig. S4), which suggested a late burst diversification in this liverwort family.

Diversification dynamics and rate shifts were slightly different when members of the cryptic species complex were included. In the BAMM phylorate in which ten taxa of A. pinguis complex were included (Fig. 3A), four shifts in speciation rates were observed at the crown nodes of Riccardia, Metzgeria, Aneura, and the clade of Riccardia pallida at around 220, 150, 80, and 45 Mya, respectively. When only one A. pinguis was included in the analysis (Fig. 3B), three speciation rate shifts were observed at 180 Mya for Riccardia, 110 Mya for Metzgeria, and 40 Mya for the Riccardia pallida clade. The shift at the crown node of the genus Aneura was not recovered in this analysis.

Effect of fossil calibration on divergence time estimation of Aneuraceae

Our results showed that the number of fossils used for calibration affected divergence time estimates and topology. The divergence times from the three-fossil calibration were consistently older than those from the single-fossil calibration, which is based on the most reliable fossil record (Feldberg et al., 2021, Table 2). Our estimates also placed the origin of the order and the family earlier than the previous estimate based on a more extensive set of liverwort species (Feldberg et al., 2014). Two reasons might explain the differences in the divergence time estimations between the current and previous studies.

First, the previous studies used a very different taxon sampling from our current study. Feldberg et al. (2014) only used one chloroplast region (rbcL) for 303 species of liverworts, only six of which came from family Aneuraceae, accounting for 1.8% of the known species, while Laenen et al. (2014) sampled one species from each of the genera in Aneuraceae. Our current study included a denser taxon sampling from the member of Aneuraceae, using data from three chloroplast regions. Moreover, these two studies were conducted before the updated checklist of Söderström et al. (2016), which increased the number of known Aneuraceae species substantially. Our results revealed that the increased number of samples impacted divergence time estimates, especially the crown node ages of Aneura and Lobatiriccardia, which were unable to obtain when using only one species per genus in the previous studies. In addition, most of previous studies used a typical Birth-Death (BD) model as a tree model, in which fossils were used only for node calibrations, whereas our current study used the fossilized birth-death (FBD) model to allow a more flexible use of fossils for both node and tip calibration. For the BD method, we could not assign Riccardiothallus devonicus as a calibration, because the age of this fossil was determined to be earlier than the age of Aneuraceae, of which it was presumed to be the member. Our results from the BD and FBD models showed small differences in age estimates, assuring that the FBD process was efficient. Therefore, the different taxon samplings have a greater effect on age estimates than the different tree priors.

Second, the uncertainties of divergence dating have existed in many groups of plants, especially when trying to estimate deep divergences in the higher taxonomic ranks. For instance, the order Brassicales has been reported to originate at 103 Mya with a range of estimation from 124.0 to 93.5 Mya, using six combinations of fossil calibration (Cardinal-McTeague et al. 2016). Even in the fossil-rich genus of Nothofagus, the age estimate of the genus varied between 113.6 to 13.3 Mya (Sauquet et al. 2012). These studies agreed that a greater amount of fossil age constraints could be helpful, but large uncertainties remained for estimations at the higher taxonomic ranks. Secondary calibration could be used to alleviate this problem, but the available estimates for bryophytes are inconsistent and were not used in this study.

Previous estimates of early land plant divergences from chronograms have been a subject of much debate, as they tend to extend into the time before the known date for the earliest land plant (475 Mya; Bowles et al., 2022; Graur & Martin, 2004; Harris et al., 2022; Morris et al., 2018). Our estimate of the origin of Metzgeriidae also experienced a similar issue of a wide HPD interval, likely due to the small number of fossils. However, the paucity of fossil records in liverworts makes it difficult to include more fossils into the estimation with some confidence, but our results from the single-fossil calibration were consistent with the known earliest land plants and likely to be the best estimates we have to date (Wellman and Gray 2000; Hernick et al. 2008). The chronogram inferred from the single-fossil calibration can be used for further analyses in biogeography or the evolution fungal associated relationships with less uncertainty than the previous estimates.

Molecular dating can also offer insights into fossil taxonomic placement. In our case, Riccardiothallus devonicus was originally assigned as a member of Aneuraceae, but Tomescu et al. (2018) questioned such an assignment based limited morphological and anatomical description of Riccardiothallus devonicus. The estimate age of R. devonicus was also earlier than any other estimates of Aneuraceae so far, supporting the placement of R. devonicus outside Aneuraceae. Our results provide an example of how molecular dating can provide evidence for fossil taxonomic reassignment. For example, the molecular data had been used to change of the placements of several squamate reptile fossils (Wiens et al. 2010). Morphological and molecular data can inform each other to help reconstruct more accurate evolutionary histories of organisms.

Placement of two monotypic genera

The current reconstruction placed the two monotypic genera (Verdoornia and Afroriccardia) differently from the previous studies. In the case of Verdoornia, Preußing et al. (2010b) and Bechteler et al. (2021) reconstructed Riccardia as the earliest diverging lineage, while our results recovered Verdoornia as the earliest divergence lineage in Aneuraceae. The differences could be the results of a denser sampling among Aneuraceae and longer alignment in the current study compared to the previous one. Our current results supported the original concept of the unique gametangia position in the genus Verdoornia in the reconstruction of Forrest and Crandall-Stotler (2004) phylogeny of Metzgeriidae.

For Afroriccardia, the current topologies were also slightly different from the reconstruction of Riccardia phylogeny (Rabeau et al., 2017), while Afroriccardia and Riccardia were recovered as the sister group with poor support value. Our topologies placed Afroriccardia comosa species as a sister to the rest of the Aneura + Lobatiriccardia clade with strong support (PP > 0.95). Afroriccardia was shared several characters with Riccardia in their female branches and the archegonia formation (Reeb and Gradstein 2020), suggesting their close relationship. However, Afroriccardia and Lobatiriccardia are also similar in their pinnately branched pattern and rhizoid expansion on the ventral side. While our results provide a stronger case for the monophyly of Afroriccardia + Aneura + Lobatiriccardia, additional characters, such as oil bodies, male thallus, gemma, and sporophyte, should be observed to help identify synapomorphies of the particular clades in Aneuraceae.

Multiple speciation rate shifts inside Aneuraceae

Within Aneuraceae, our BAMM results showed a significant rate shift at the origin of Riccardia (at 220 Mya) and another inside the genus (R. pallida clade at 45 Mya). The evidence of the R. pallida clade was also observed in Rabeau et al. (2017) and Preußing et al. (2010b), who referred to this clade as the “Andean clade” for their primary distribution in the neotropical Andes. The increased diversification of the Andean clade might be the result of the climate change in the Middle-Late Eocene Cooling until Early Oligocene, about 45–30 Mya. During this event, the global temperature decreased substantially, even in the tropical zone, to as low as 24.4˚C in the Northern South America across the season (Golonka et al. 1994; Crowley 2012; Scotese et al. 2021). This period also coincided with the formation of Central and Northern South America (http://www.scotese.com). Simultaneously, the lithological study on paleoclimate also indicated that this region used to be a warm temperate zone prior to the Oligocene (Boucot et al. 2013). Therefore, Central America and Tropical South America may have become a hyper-diverse region for Riccardia as new ecological opportunities emerged during the geological and climatic changes in the Middle-Late Paleogene.

Another possibility of increased speciation in Riccardia may have to do with asexual reproduction (e.g., gemmae). Within the family, Riccardia is the only genus that can produce gemmae and display the shifts from the BAMM phylorate results. Producing asexual propagules may be a good strategy for dioicous bryophytes to spread and colonize in various environments and habitats, as it is more cost-effective than reproducing sexually through sporophytes (Miller 1982; Fuselier 2008; Devos et al. 2011; Shaw et al. 2011; Kraichak 2012; Holá et al. 2014). Newton and Mishler (1994) pointed out the connection between asexual reproduction and genetic diversity in mosses. In an asexual population of bryophytes, the mutation in the vegetative cell may occur in a single apical cell at a shoot or the sporophyte, and these cells can also pass on and accumulate the mutations through generations. Therefore, when a population with asexual reproduction exists at the margin of the distribution or at the boundary of fragmentation, then allopatric speciation (i.e., mountain range or terrain or islands) could occur from that population with effect of habitat and microhabitat (Mishler 1988; Buczkowska et al. 2010; Frey and Kürschner 2011; Flagmeier et al. 2013; Laenen et al. 2016).

Diversification of the simple thalloid liverworts: Symbiosis or Climate

Symbiosis with fungi was hypothesized to be the key to the successful colonization of land plants, including species of Aneuraceae. Krause et al. (2011) showed that within the liverworts, the Tulasnellales fungi (Basidiomycota) could be found only in Aneuraceae. However, for several reasons, fungal symbioses within liverworts appear to be different from that of vascular plants. First, the fungi colonization in a liverwort thallus depends more on ecological factors than the host’s identity (Bidartondo and Duckett 2010). Second, the relationship between Aneuraceae and their fungi is often considered a “secondary symbiotic acquisition” because the fungal association within Metzgeriidae had disappeared from all families, except for Aneuraceae. Within Aneuraceae, subsequent losses were found in the genus Riccardia (Preußing et al. 2010b). After that, the fungal association reappeared again in the leafy liverwort clade (Jungermanniidae) and most other land plants (Rimington et al. 2018, 2020; Pressel et al. 2021). It is likely that fungal associations evolved multiple times within bryophyte clades and could be linked to diversifications of various groups, including Aneuraceae.

While arbuscular mycorrhizal fungi (AMF) have been demonstrated to enhance the fitness of some liverwort species (Humphreys et al. 2010), the effect of fungal association on diversification within Metzgeriidae is less clear. Members of Metzgeriidae revealed a wide range of fungal associations from fully myco-heterotrophic like Cryptothallus to plants completely free of fungi, e.g., in Metzgeria and Riccardia, with a few exceptions of three endemic species to New Zealand (Pressel et al., 2010). Our results showed an increased diversification at the ancestral node of Aneuraceae. It is possible that the increased diversification of Aneuraceae was associated with fungal symbiosis, while the other two rate shifts in non-symbiotic clades could be the results of the other innovations (Preußing et al. 2010b).

One possible explanation from both chronogram and LTTs results is that novel climatic conditions allowed the radiation Aneuraceae ancestor. Our chronogram placed the origin of the family Aneuraceae at the beginning of Carboniferous. During this period, the first canopy was formed, decreasing temperature, and increasing humidity from the shades of vascular plants. This combination was an optimal condition for simple thalloid liverworts, as we can observe in the niche of extant Aneuraceae species in shaded areas with high moisture. This period coincided with the formation of the southern hemisphere with cool-temperate to periglacial climate through Late Carboniferous. It is possible that cooling climate could contribute the diversification of Aneuraceae, but a detailed analysis on ancestral area reconstruction and fossil records of this group will be required to test this particular hypothesis (DiMichele et al. 2001; López-Gamundí and Buatois 2010).

Effect of cryptic species complex on macroevolutionary analysis

Most of the current macroevolutionary analyses rely on the assumption that the operational taxonomic units (OTUs) are clearly defined, which subsequently do not accommodate taxonomic groups with cryptic species. In the current study, we confirmed a case of cryptic species within the genus Aneura, similar to other previous studies (Wickett and Goffinet 2008; Wickett et al. 2008; Bączkiewicz et al. 2017). Wickett et al. (2008) detected rapid mutations and significant deletions in the plastid genome of Aneura species, suggesting high diversification within the genus. Moreover, many studies revealed that A. maxima and A. mirabilis are nested within the A. pinguis complex, making A. pinguis a paraphyletic group. Even within the A. pinguis complex, ten putative species were detected using a phylogenetic species concept (Bączkiewicz et al., 2017). In this study, we performed BAMM using one A. pinguis sequence and ten sequences to represent the putative cryptic species.

In our case, the inclusion of ten putative species of A. pinguis resulted in an additional rate shift within the Aneura clade (Fig. 3A) compared to the analysis with only one A. pinguis sequence. Previous studies in other taxonomic groups found similar impacts of cryptic species on the BAMM analysis. For example, Liu et al. (2018) assigned all the 43 cryptic species of Asian frogs to the chronogram and BAMM speciation analysis and detected the shifts within the genus Megophrys and evolutionary radiation of the Panophrys group, which contained primarily cryptic species. Another example is a study on passerine birds, in which BAMM can detect a high speciation rate inside these cryptic species, suggesting adaptive radiation in this group (Mason et al., 2016). Mamos et al. (2021) identified eight molecular operational taxonomical units (MOTUs) from a species complex of amphipods (Gammarus balcanicus) with species delimitation approaches. They used these MOTUs instead of the traditional species in the BAMM analysis and could detect one shift inside this cryptic species and reveal the new species. The current and previous studies demonstrated that the omission of cryptic species could underestimate shifts occurring within a phylogeny. Therefore, a clear definition of taxonomic units and empirically delimiting species boundaries are critical components of current macroevolutionary analyses.

Acknowledgments

We would like to thank Matthew P. Nelsen from the Field Museum, Chicago, IL, for his recommendation about Bayesian Inference analyses, packages, and tools in the BEAST environment. In Defense of Plants’s podcast, their episode with Alexander C. Bippus from Oregon State University, Corvallis, OR, about the bryophyte fossils which inspired me, fulfilled my discussion perspective, and made confidence to this paper.

Funding

N. Anantaprayoon’s graduate program was supported by the Graduate Program Scholarship from the Graduate School, Kasetsart University. This project was financially supported by the Office of the Ministry of Higher Education, Science, Research, and Innovation, and the Thailand Science Research and Innovation through the 2021 Kasetsart University Reinventing University Program, Thailand, and by National Research Council of Thailand (NRCT), 2022 annual funding under Thailand Science Research and Innovation (TSRI).

Author Contributions

N. A. designed, performed formal analyses, prepared figures, and drafted the manuscript. P. W. and S. D. L. had provided conceptualized, suggestions, and revised this manuscript. E. K. designed, conceptualized, provided support for data analyses, and revised the manuscript. All authors have reviewed and approved this manuscript.

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Evolution of the most species-rich family of simple thalloid liverworts (Aneuraceae): a time-calibrated perspective into its evolutionary history and diversification

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­Evolution of the most species-rich family of simple thalloid liverworts (Aneuraceae): a time-calibrated perspective into its evolutionary history and diversification

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Evolution of the most species-rich family of simple thalloid liverworts (Aneuraceae): a time-calibrated perspective into its evolutionary history and diversification