Multiple colonization and dispersal events hide the early origin and induce a lack of genetic structure of the moss Bryum argenteum in Antarctica

Abstract The dispersal routes of taxa with transoceanic disjunctions remain poorly understood, with the potential roles of Antarctica not yet demonstrated. Mosses are suitable organisms to test direct intra‐Antarctic dispersal, as major component of the extant Antarctic flora, with the cosmopolitan moss Bryum argenteum as ideal target species. We analyzed the genetic structure of B. argenteum to provide an evolutionary time frame for its radiation and shed light into its historical biogeography in the Antarctic region. We tested two alternative scenarios: (a) intra‐Antarctic panmixia and (b) intra‐Antarctic genetic differentiation. Furthermore, we tested for evidence of the existence of specific intra‐Antarctic dispersal routes. Sixty‐seven new samples (40 collected in Antarctica) were sequenced for ITS nrDNA and rps4 cpDNA regions, and phylogenetic trees of B. argenteum were constructed, with a focus on its Southern Hemisphere. Combining our new nrDNA dataset with previously published datasets, we estimated time‐calibrated phylogenies based on two different substitution rates (derived from angiosperms and bryophytes) along with ancestral area estimations. Minimum spanning network and pairwise genetic distances were also calculated. B. argenteum was potentially distributed across Africa and Antarctica soon after its origin. Its earliest intra‐Antarctic dispersal and diversification occurred during a warming period in the Pliocene. On the same timescale, a radiation took place involving a dispersal event from Antarctica to the sub‐Antarctic islands. A more recent event of dispersal and diversification within Antarctica occurred during a warm period in the Pleistocene, creating favorable conditions also for its colonization outside the Antarctic continent worldwide. We provide evidence supporting the hypothesis that contemporary populations of B. argenteum in Antarctica integrate a history of both multiple long‐range dispersal events and local persistence combined with in situ diversification. Our data support the hypothesis that B. argenteum has been characterized by strong connectivity within Antarctica, suggesting the existence of intra‐Antarctic dispersal routes.

of the existence of specific intra-Antarctic dispersal routes. Sixty-seven new samples (40 collected in Antarctica) were sequenced for ITS nrDNA and rps4 cpDNA regions, and phylogenetic trees of B. argenteum were constructed, with a focus on its Southern Hemisphere. Combining our new nrDNA dataset with previously published datasets, we estimated time-calibrated phylogenies based on two different substitution rates (derived from angiosperms and bryophytes) along with ancestral area estimations. Minimum spanning network and pairwise genetic distances were also calculated. B. argenteum was potentially distributed across Africa and Antarctica soon after its origin. Its earliest intra-Antarctic dispersal and diversification occurred during a warming period in the Pliocene. On the same timescale, a radiation took place involving a dispersal event from Antarctica to the sub-Antarctic islands. A more recent event of dispersal and diversification within Antarctica occurred during a warm period in the Pleistocene, creating favorable conditions also for its colonization outside the Antarctic continent worldwide. We provide evidence supporting the hypothesis that contemporary populations of B. argenteum in Antarctica integrate a history of both multiple long-range dispersal events and local persistence combined with in situ diversification. Our data support the hypothesis that B. argenteum has been characterized by strong connectivity within Antarctica, suggesting the existence of intra-Antarctic dispersal routes.

| INTRODUC TI ON
Some plant species exhibit transoceanic disjunctions and, for species with disjunct distributions, their underlying dispersal routes and diversification patterns across space and time remain to be clarified. To explain the disjunct distributions of austral plants between South America and the western Pacific, Winkworth, Hennion, Prinzing, and Wagstaff (2015) hypothesized both out-of-Antarctica and intra-Antarctic dispersal routes, identifying two potential periods warmer than today that could have supported such dispersal (Miocene,and Pliocene,. However, while the potential for dispersal from the Antarctic continent is recognized, the identification of taxa that followed this pattern remains challenging due to the subsequent extinction of most of the Antarctic biota. Indeed, in most cases topologies based on, for instance, extant plant taxa are largely uninformative with respect to dispersal routes due to the fragmentation of the extant flora or to potentially incomplete sampling (Winkworth et al., 2015).
Among bryophytes, Bryum argenteum Hedw. is a cosmopolitan moss, characterized by global distribution from tropical to polar latitudes, from sea-level up more than 4,000 m altitude, and from pristine natural environments to heavily human-impacted habitats (Bansal & Nath, 2013;Ingimundardottir, Weibull, & Cronberg, 2014;Kurschner, 1996;Puche & Gimeno, 2000). In Antarctica, B. argenteum has among the widest geographical distribution and highest abundance of the mosses present (Ochyra et al., 2008), thereby being an ideal model species to infer colonization routes and dispersal patterns. The historical biogeographic and recent demographic patterns of some Antarctic biota may have been facilitated by in situ persistence in refugial areas associated with geothermal activity, such as those located in the northern Antarctic Peninsula and Scotia Arc, Marie Byrd Land, and Victoria Land (Convey & Smith, 2006;Fraser, Terauds, Smellie, Convey, & Chown, 2014).
Recently, molecular phylogeographic studies have been applied to reconstruct the global distribution of B. argenteum, including its Antarctic range wherein the occurrence of multiple colonization events and in situ persistence have been proposed (Hills, Stevens, & Gemmill, 2010;Skotnicki, Mackenzie, Clements, & Selkirk, 2005), and higher genetic variation reported than in all other regions globally (Pisa et al., 2014). Building upon the multiple colonization event scenario proposed by Pisa et al. (2014) and using a newly enlarged dataset for Antarctica, in the current study we aim to clarify understanding of the historical biogeography of this species in the Antarctic continent and the surrounding regions.
To provide an evolutionary timeframe for the inter-and intra-Antarctic dispersal and radiation events for B. argenteum, and to reconstruct its historical biogeography in this region, we generated time-calibrated phylogenies based on two different models of substitution rates, inferred, respectively, from the Moss Tree of Life (Laenen et al., 2014) and from a broad meta-analysis of molecular substitution rates in angiosperms (Kay, Whittall, & Hodges, 2006).
More specifically, we assessed which of the following scenarios best explain the Antarctic population genetic structure and historical biogeography of B. argenteum: Scenario I: There is no evidence of genetic structure, pointing to panmixia and/or strong connectivity across Antarctica; Scenario II: There is strong genetic structure that matches the geographic pattern of Antarctic biogeographic regions, known as Antarctic Conservation Biogeographic Regions (ACBRs), for the Antarctic Peninsula (North-West Antarctic Peninsula-NWAP; North-East Antarctic Peninsula-NEAP; and Central-South Antarctic Peninsula-CSAP) and for part of Continental Antarctica (Northern Victoria Land-NVLCA; Southern Victoria Land Continental Antarctica-SVLCA) (see Terauds et al., 2012;Terauds & Lee, 2016).
Furthermore, we hypothesized that patterns of genetic structuring would correspond to specific intra-Antarctic dispersal routes, as suggested by Winkworth et al. (2015), in particular along the Transantarctic Mountains and/or the coast of continental Antarctica, with dispersal events occurring during periods of reduced ice cover (Cannone, Convey, & Guglielmin, 2013).

TA B L E 1 (Continued)
1994 and 2003 by PNRA (Programma Nazionale Ricerche in Antartide) and BAS (British Antarctic Survey) researchers (see Table 1). Samples were collected along two latitudinal transects.
Eleven samples were obtained from key geographic areas in the AP, North America and seven from Asia) and the Southern Hemisphere (seven from Africa and eight from South America; Figure 1a; Table 1).

| Molecular data
We analyzed the internal transcribed spacer (nrITS) region and the chloroplast gene rps4 (cpDNA) to obtain data comparable with the available literature. From each collected gametophyte, total genomic DNA was extracted using the PowerPlant DNA Isolation Kit (Qiagen, Inc.) and Proteinase K digestion. PCR amplification of the ITS region was carried out using primers specifically redesigned for B. argenteum (Pisa et al., 2014), while rps4 was amplified using the primers RPS5 (Nadot, Bittar, Carter, Lacroix, & Lejeune, 1995) and TRNAS (Buck, Goffinet, & Shaw, 2000). Each PCR reaction contained 10 μL reaction volume with 0.2 μM concentration of each primer, 5 μl QIAGEN Multiplex (QIAGEN multiplex PCR kit), and 2 μl extracted DNA. Both amplifications were performed using a thermocycler (BIO RAD -T100 Thermal Cycler) with the following protocol: 15 min at 95°C followed by 40 cycles each of 94°C for 30 s, 56°C for 90 s and 72°C for 90 s, and finally 10 min at 72°C.
PCR products were purified with EuroSap enzymes (Euroclone) and directly sequenced in both forward and reverse directions (MACROGEN Inc. Amsterdam, The Netherlands; http://www.macro gen.org) using a 3730XL DNA Sequencer. The sequences were aligned using Bioedit ver.7.0.9 (Hall, 1999) and edited by eye allowing gaps where necessary to conserve homology among sequences.
All the sequences generated in this study, deposited in GenBank under the accession numbers KT947989-KT948009, MF152769-MF152780, MK234228-MK234295 and MG651608-MG651645 (Table 1), were grouped into haplotypes using DnaSP ver. 5.10 (Librado & Rozas, 2009). The nrITS region has been more extensively analyzed than the cpDNA region in B. argenteum, and we therefore focused on this specific nuclear marker for the biogeographical analyses since it provides a more comprehensive biogeographical context. The nrITS sequences obtained in the present study (67) were aligned with all globally available data for the species, which gave a total of 149 nrITS sequences (Hills et al., 2010;Pisa et al., 2014;Rankin et al., 2017;Skotnicki et al., 2005; Table S1). Hereafter, we refer to this alignment as the global nrITS dataset.

| Phylogenetic analyses
The phylogeny of B. argenteum was independently reconstructed using the nrITS and cpDNA datasets obtained in the present study ( work on the genus Bryum s.l. (Hills et al., 2010;Pisa et al., 2014;Skotnicki et al., 2005). Maximum likelihood (ML) analyses were performed using the GTR + G and HKY + I replacement models of nucleotide evolution for nrITS and cpDNA, respectively, as recommended by jModelTest software (Posada, 2008) selected under the Akaike information criterion. Confidence values of the nodes were estimated using 1,000 bootstrap replicates. The analyses were computed using PAUP 4.0b (Swofford, 2002) and GARLI ver. 1.0 (Bazinet, Zwickl, & Cummings, 2014;Zwickl, 2006).  estimated clade ages that were congruent (i.e., overlap). For each analysis, Bayesian reconstructions were conducted using four different tree priors, including Yule and birth-death process priors, as well as coalescent priors such as the models Constant Size and Bayesian Skyline. For each analysis, we conducted two independent runs to ensure convergence of the MCMC. We then compared their posterior distributions using the marginal likelihood estimate (MLE) of each model, estimated from stepping-stone sampling (SS) and path sampling (PS). We performed MLE using SS with 150 path steps, each with a chain length of one million iterations, and the other parameters were set by default. We directly calculated the log-Bayes factors from MLEs and used BF to compare the support of all the models tested. We considered BF values above 2 to indicate that one model was significantly favored over another. In both cases (calibrations I and II), results from PS and SS MLEs were very similar, and a constant size tree model under an uncorrelated relaxed molecular clock model was selected (see Table S2).

| Timeframe and ancestral area estimation
To provide evidence of inter-and intra-Antarctic biogeographic range evolution, we used the R package BioGeoBEARS (BioGeography with Bayesian Evolutionary Analysis in R Scripts) (Matzke, 2014) to perform ancestral area estimations based on the two time-calibrated MCC trees obtained from BEAST (calibrations I and II) (see Table S3). After removing the outgroups, we performed the ancestral area estimations across the ingroup. We applied three different biogeographical models (DEC, DIVALIKE, and BAYAREA) under a maximum likelihood framework and compared how well they fitted the data using the Akaike information criterion corrected for small sample size (AICc).

| Molecular indices and genetic structure of samples from Antarctic regions
The population genetic structure of B. argenteum was assessed by specifically using a restricted multiple alignment of 40 new sequences collected in Antarctic regions (from 63° to 77°S) plus a further 29 sequences available from the literature which also included the sub-Antarctic islands (Hills et al., 2010;Pisa et al., 2014;Rankin et al., 2017;Skotnicki et al., 2005), giving an overall latitudinal range from 46° S to 78° S (Table S1). This multiple alignment covered the AP (n = 15), CA (n = 48), four sequences from sub-Antarctic islands (SAI) at 46° S (Prince Edward Island, Crozet Islands and Possession Island) and two from the northern Maritime Antarctic South Sandwich Islands (MASSI) at 57° S (Candlemas Island) (see Table S1).
For visual inspection, a minimum spanning network (MSN) was created using statistical parsimony criteria as implemented in TCS ver. 1.18 (Clement, Posada, & Crandall, 2000). Descriptive statistics of haplotype and nucleotide diversity were obtained using DnaSP and Arlequin ver. 3.5.1.3 (Excoffier & Lischer, 2010). Estimated values for the entire Antarctic dataset, and for AP and CA considered separately, included the number of sequences (N), number of haplotypes (n), nucleotide diversity (π), and haplotype diversity (H). Levels of pairwise genetic differentiation between the Antarctic regions (i.e., CA and AP) and between local biogeographic classification (i.e., NEAP, NWAP, and CSAP for AP; and NVLCA and SVLCA for CA) were estimated as pairwise genetic distances (Φ ST ), and their significance tested by 1,000 random permutations. Among the 67 nrITS sequences, 34 haplotypes were identified and nine of these were found in Antarctic regions (Table 1).
In both phylogenetic trees (based on nrITS and cpDNA sequences), the Antarctic samples were not clustered in monophyletic clades (Figure 2a, b). The nrITS tree topology showed polytomy between Antarctic sequences (Figure 2a), while the cpDNA tree had a more complex topology in which Antarctic samples were distributed in three clades (pp> 0.9) (Figure 2b) (See Table 1).
The nrITS phylogeny tree of Antarctic samples (Figure 2a) was resolved analyzing the global nrITS dataset (see Table S1). In particular, the global nrITS phylogenetic tree identified four statistically sup-  Table S2). The geographic distribution of haplotypes is detailed in Table 1 and Table S1. Results of ancestral areas analysis based on DIVALIKE biogeographical model (see Table S3) in relation to the BEAST chronogram are also indicated. Posterior probability values at nodes (pp> 0.90) are indicated. The 95% highest posterior density (HPD) intervals are provided in Table 2

| Timeframe and ancestral area estimation
The phylogenetic reconstructions based on the two different schemes of substitution rates obtained from angiosperms (calibration I in Fig. S1) and mosses (calibration II in Figure 3) provided a congruent timeframe (see Table 2), dating the species' origin at 15.4 Ma (95% HPD: 21.0-10.7 Ma; pp = 1) or 14.9 Ma (95% HPD: 22.6-9.6 Ma; pp = 1), respectively. The first lineage split within the species was inferred to have occurred around 5.7 Ma (95% HPD: 7.7-4.0 Ma; pp = 1) or 5.9 Ma (95% HPD 9.0-3.8 Ma; pp = 1), respectively. The ancestral area analyses using the two BEAST chronograms selected DIVALIKE as the best-fit model (Table S3). Both analyses indicated that the most recent common ancestor of B. argenteum was potentially distributed across Africa and Antarctica, although the calibration I analysis also indicated its presence in Asia and North America (See Table 2, Figure 3; Fig. S1). Both dating approaches generated closely similar timeframes for the diversification of each of the four clades, although the more recent internal splits (A vs. B and C vs. D) were not significantly supported (pp < 0.8). Following the calibration I and calibration II schemes, the clade divergence events were dated as follows (Table 2 Figure 3; Figure S1). Clade D showed an African origin in both chronograms, but a North American origin was also inferred in the calibration I chronogram (Table 2, Figure 3; Figure S1).

| Intra-Antarctic genetic diversity
The species' phylogeographic structure within Antarctica is illustrated in Figure 4. A star-like structure was clear around the central position of Hap1, present and widespread in AP and CA, from which six haplotypes radiated with a single mutational step and one haplotype (Hap7) with two mutational steps. A second haplotype (Hap10) was at the center of a similar pattern, from which radiated Hap11 and Hap64 (located in CA) and Hap9 and Hap26 (located in AP). Both South Sandwich Islands (MASSI, Candlemas I.) and Sub-Antarctic islands (SAI; Prince Edward I., Crozet I. and Possession I.) radiated from Hap10 with between one (Hap14) and seven (Hap 13) mutational steps (Figure 4). There were six mutational steps between Hap10 and Hap1, giving the overall network a dumbell shape (Figure 4).
Haplotype (H) and nucleotide (π) diversities in Antarctica were 0.54 and 0.025, respectively, while the H and π values in AP and CA ranged between 0.46 and 0.73 and between 0.023 and 0.033, respectively (Table 3). Pairwise Ф ST comparisons between the two Antarctic regions were not significant.
Analyzing genetic differentiation separately within CA and AP, significant Ф ST values (p < .05) were only recorded in the North-West Antarctic Peninsula (NWAP) (see Table 3). This area was significantly  Table 4).

| Phylogeny of B. argenteum
The phylogenetic tree and biogeographic evidence, achieved by comparing dating analyses using two different calibration schemes The molecular biogeographic footprint of clade D suggests that Antarctic mosses may have experienced geographic and genetic isolation, plausibly linked to cooling events that occurred in the late Pliocene and early to mid-Pleistocene (Head & Gibbard, 2015) that likely erected climatic barriers. From the ancestral haplotype of clade D (Hap10), we identified several "private" haplotypes that appear to have radiated in situ in both the AP and CA. Members of this clade are also present on the SAI, sharing Hap10 as a common ancestor. This supports a hypothesis of colonization from the AP or CA, and is consistent with evidence from Sanionia uncinata (Hedenäs, 2012) and the bipolar moss species Polytrichum juniperinum (Biersma et al., 2017). We recognize it is possible that some Antarctic biota could have survived on the sub-Antarctic islands (particularly South Georgia) or volcanic South Sandwich Islands during glacial periods, from where they may have then recolonized continental Antarctica during warmer interglacials (Rogers, 2007); however, there is very little evidence available supporting this for terrestrial biota, other than for the South Sandwich Islands  Table 1 and Table S1 Continental Antarctica  Table 1 and is currently only identified from a heavily glaciated area of the Clade B was inferred to have diverged around 1.2-1.3 Ma (see Table 2), probably exploiting favorable conditions that occurred during multiple short interglacial periods in the Pleistocene (for instance one estimated at 1.07 Ma; Swanger, Marchant, Schaefer, Winckler, & Head, 2011). Hap1 may have originated during this specific interglacial, with wide dispersal also being favored by partial collapse of Antarctic ice sheets (Pollard & DeConto, 2009;Swanger et al., 2011), and favorable conditions for the species' dispersal and colonization outside the Antarctic continent worldwide (see Table S1). Subsequent periods of cooling and glacial maxima may again have resulted in isolation, promoting in situ radiation from the widespread Hap1, consistent with the star-like Antarctic haplotype network of this clade.

Antarctic Peninsula
The complex history of Antarctic colonization here recorded in B. argenteum and its persistence through glacial periods within the continent complements recent observations in multiple groups of terrestrial biota (Biersma et al., 2017;Convey et al., 2008Convey et al., , 2018Convey et al., , 2020McGaughran, Tearuds, Convey, & Fraser, 2019), including many invertebrate groups, lichens, and other mosses that currently occur in habitats and areas that are also commonly characterized by B. argenteum. These important components of the Antarctic biota must therefore have had environmentally suitable refugia available to them (Convey et al., , 2020. However, the precise locations of such refugia remain unknown , as does the degree of connectivity or isolation between individual locations.

| Intra-Antarctic genetic structure and dispersal routes
In a harsh climatic context such as that of Antarctica, genetic bottlenecks might be expected as a result of colonization events followed by geographical isolation and survival without demographic expansion, likely leading to biogeographic differentiation at an intra-Antarctic scale. However, although limited by relatively small sample sizes and intra-Antarctic coverage, our data indicate limited genetic differentiation between the AP and CA, providing some evidence for panmixia within Antarctica for this moss species and at least partially supporting our initial Scenario I.
Our analyses also provide support for the existence of intra-Antarctic dispersal routes between the Antarctic Peninsula and Continental Antarctica, which could contribute to the overall lack of Antarctic population genetic structure in B. argenteum. Lack of evidence supporting intra-Antarctic genetic differentiation could be related to the occurrence of intra-Antarctic dispersal events following colonization, admixing the genetic differentiation signal provided by older and region-specific colonization events. Although some basal haplotypes are restricted exclusively to the AP region, the widespread distribution of Hap1 and the lack of genetic structure among the ACBRs support the existence of connection by dispersal routes between Victoria Land (CA) and the AP (Cannone et al., 2013;Ochyra et al., 2008). Other moss species such as Ceratodon purpu- TA B L E 4 Pairwise Ф ST comparisons among the ACBRs recognized in the Antarctic Peninsula (AP) and in Continental Antarctica (CA) and their significance after 1,000 permutations, indicated as follows: * p < .05; ** p < .01. Geographic regions are as given in Table 1 primarily in the AP and Victoria Land (Cannone et al., 2013;Ochyra et al., 2008). This species has also been reported from three widely separated coastal sites around continental Antarctica, in Marie Byrd Land, Dronning Maud Land, and Enderby Land (Ochyra et al., 2008

| Geographic distribution of Antarctic exclusive haplotypes
Antarctic exclusive haplotypes (Table 1 and have played a role in the longer-term regional persistence of B. argenteum, but indicated that more investigations were needed. Although, as noted above, geothermal areas associated with volcanic activity may have favored the persistence of ice-free ground locally, providing refugia for some terrestrial biota in Antarctica (Convey & Smith, 2006;Fraser et al., 2014), our data indicate that the geothermal refugia hypothesis may not be the most appropriate hypothesis to apply to B.
argenteum, as previously envisaged by Ochyra et al. (2008). Indeed, assuming niche conservatism (the tendency of species to retain their niches and related ecological traits over time; Wiens et al., 2010), the contemporary distribution of B. argenteum in Antarctica shows no close relationship to geothermal sites. In Victoria Land, the species is absent from Mount Melbourne fumaroles (also Mount Rittmann) (Bargagli, Broady, & Walton, 1996) and there is one specimen record from 1965 (Ochyra et al., 2008) and one more recent sequence record (98% similarity) in an eDNA study (Fraser, Connell, Lee, & Cary, 2018) for the Mt. Erebus area. The species is specifically recorded on Deception Island in the maritime Antarctic, although this latter location has a relatively recent age, originating only some tens of thousands of years ago (Smellie, 1988 et al., 2015). Given niche conservatism, as the contemporary distribution of B. argenteum includes elevations up to 1,050 m in Victoria Land (Ochyra et al., 2008), the survival of this species in nunatak refugia in this region can be hypothesized.
Long-term in situ persistence of B. argenteum in Antarctica could have been achieved both in refugia or, potentially, through cryptobiosis (long-term survival in a biologically inactive state). An example of this has been demonstrated recently at Rothera Point (AP), where clump-forming mosses survived using cryptobiosis over six centuries of cold-based glacier burial and, after re-exposure due to glacier retreat, were able to return to a metabolically active state (Cannone et al., 2017). A similar inference was made by La Farge, Williams, and England (2013) in the Canadian High Arctic, as well as by Roads, Longton, and Convey (2014) at Signy Island (South Orkney Islands) within bank-forming mosses over a period of up to two millennia preserved in permafrost. In Victoria Land, cold-based glaciers were widespread between c. 5-7 Ma and 2.5 Ma (Di Nicola et al., 2012;Smellie et al., 2014), providing the possibility of survival during glaciations through cryptobiosis, although it has to be accepted that the duration of glacial periods (tens to hundreds of thousands of years) is considerably longer than any currently available demonstration of cryptobiotic survival other than in microorganisms (Convey et al., 2020).
In conclusion, this study has provided evidence that B. argenteum occurred in Antarctica and Africa (as well as possibly Asia and North America) soon after the species' origin and before the differentiation of its major constituent clades. Its earliest intra-Antarctic dispersal and diversification occurred during Pliocene, likely favored by paleoclimatic warming during this era, with this radiation also leading to colonization of the sub-Antarctic islands (around 46°S latitude) through migration from Antarctica. More recent dispersal and diversification within Antarctica also likely occurred during warm interglacial periods in the Pleistocene, a geological period that most likely shape its extant worldwide distribution.
Contemporary diversity in Antarctic B. argenteum has been influenced both by multiple long-distance dispersal events and by local persistence and in situ diversification, determining its population genetic structure within the region. Our analyses confirm that B.
argenteum shows strong intra-Antarctic connectivity (scenario I), rejecting the hypothesis that genetic structure within this species would match the distribution of Antarctic Conservation Biogeographic Regions (scenario II). The lack of genetic differentiation between AP and CA may be explained by the existence of intra-Antarctic dispersal routes at earlier times.

ACK N OWLED G M ENTS
The

CO N FLI C T O F I NTE R E S T
All authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All molecular data are deposited in Genbank and the accession numbers of original sequences produced in this study are reported in Table 1.