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Biogeography and Systematics of the Genus Axyris (Amaranthaceae s.l.)

Axyris is a small genus of six species with a disjunct geographic range. Five species are present in Siberia, Central Asia, the Himalayas, and Tibet, whereas Axyris caucasica has been registered in the Central Caucasus only. Axyris species diversity is the highest in the Altai Mountains (four spp.), followed by the Tian Shan and Pamir Mountains (three spp.), and the Himalayas and Tibet (two spp.). Axyris sphaerosperma, sometimes considered endemic to Southern Siberia, in fact has a disjunct range: it is present in the lowlands of Eastern Siberia and in the Altai, Tian Shan, and Pamir Mountains. It has also been found in Mongolia and China for the first time. An updated detailed distribution of Axyris in Siberia is presented on the basis of thorough herbarium revisions. One nuclear and three plastid markers were selected for phylogenetic analysis. Divergence times were estimated using a time-calibrated Bayesian approach. Axyris shows two major clades: an Axyris amaranthoides clade and a clade including the remaining species. The latter clade consists of two subclades (A. sphaerosperma/A. caucasica and A. mira/A. prostrata + A. hybrida). The crown age for Axyris dates back to the Early Pliocene (~5.11 mya, the Zanclean). The ancestral range of Axyris covers Southern Siberia, Mongolia, NW China, and the Tian Shan/Pamir Mountains, with extensions toward Eastern Siberia, the Himalayas/Tibet, and the Caucasus. Fruit and seed characteristics of Axyris are discussed with reference to the present phylogenetic results. Closely related A. sphaerosperma and A. caucasica have the thickest seed coat among all Chenopodiaceae, and these traits have probably evolved as adaptations to extremely low winter temperatures. This reproductive peculiarity may explain the disjunct range of A. sphaerosperma, which is restricted to harsh climatic conditions.


Introduction
Axyris L. is a small genus of Chenopodiaceae Vent. s.str. (Amaranthaceae Juss. s.l.) comprising six species [1]. All of them share an annual life form, stellate pubescence of the stem and leaves, unisexual flowers, and one-seeded indehiscent fruits, usually with ear-like outgrowths in their upper part originating from the pericarp [2]. Although the genus is easily recognizable in the field, identification at the species level has often led to confusion due to the absence of reliable diagnostic characteristics. Three species-Axyris amaranthoides L., A. hybrida L., and A. sphaerosperma Fisch. & C.A.Mey.-have often been misidentified owing to their upright stems, similar leaf shapes, and overlapping geographic ranges in some parts of temperate Asia. Diagnostic methods for each species were greatly improved only recently, and pubescence details of stems and leaves and specific features of fruits and seeds appear to be the key discriminatory traits [1,2]. All Axyris species produce both heterocarpous and heterospermous diaspores, and morphoanatomical traits of fruit types are now regarded as diagnostic at the specific level [1,2].
features of fruits and seeds appear to be the key discriminatory traits [1,2]. All Axyris species produce both heterocarpous and heterospermous diaspores, and morphoanatomical traits of fruit types are now regarded as diagnostic at the specific level [1,2].
Axyris is the type genus of the tribe Axyrideae G. Kadereit & Sukhor. [13]. The genus seems to be monophyletic, judging by two or three species included in a phylogenetic analysis [13][14][15], but infrageneric relationships have not been elaborated so far. The aims of the present study were (i) to reveal the main diversity centers of Axyris, (ii) to construct an Axyris phylogeny comprising all the species of the genus and to revise the systematics of the genus, and (iii) to trace biogeographical history and radiation of Axyris on the basis of the phylogenetic analysis.

Species Distribution of Axyris in Asia
We revised the distribution of Axyris in Siberia, and in particular, we found that the range of A. sphaerosperma is drastically different from the one previously reported . Based on our recent results and the previous study [1], the highest species diversity of Axyris is seen in Southern Siberia, Mongolia,and NW China (Figure 5), where four species are present (A. amaranthoides, A. hybrida, A. prostrata, and A. sphaerosperma), followed by the Tian Shan and Pamir Mountains (A. hybrida, A. prostrata, and A. sphaerosperma) and the Himalayas/Tibet (A. mira and A. prostrata). One native species each is present in Eastern Siberia (Sakha [Yakutiya] Republic) and in the Greater Caucasus: A. sphaerosperma and A. caucasica, respectively.

Dated Molecular Phylogeny of Axyris
The combined dataset of all four markers (ITS, rbcL coding gene, atpB-rbcL and trnL-F intergenic spacers) comprises 2588 aligned bp and 21 accessions. ML and Bayesian analyses revealed identical topologies. Axyris is monophyletic with high support and it is recovered as a sister clade to Krascheninnikovia and Ceratocarpus ( Figure 6; BSL 100; PP 1). Axyris amaranthoides is resolved as sister to the rest of the species (BSL 91; PP 1). Axyris caucasica and A. sphaerosperma form a clade (BSL 96; PP 1) sister to a well-supported clade (BSL 82; PP 0.95) consisting of A. mira / A. prostrata + A. hybrida.
The diversification of Axyris started in the late Eocene ca. 25.65 mya (stem age, 95% HPD: 36.3-14.97). The crown age of Axyris dates back to ca. 5.11 mya (95% HPD: 10.91-2.82 mya) which suggests that the genus originated during the early Pliocene (the Zanclean stage). Figure 6. The maximum clade credibility tree of Axyris obtained by the BEAST2 analysis was subjected to secondary calibrations (see Methods). Posterior probabilities resulting from the Bayesian analysis are indicated above branches (only values ≥ 0.9), and the numbers below branches refer to bootstrap values resulting from the ML analysis (only values ≥ 70). Mean divergence times (values at some nodes) are shown with their 95% HPD (blue bars).

Dated Molecular Phylogeny of Axyris
The combined dataset of all four markers (ITS, rbcL coding gene, atpB-rbcL and trnL-F intergenic spacers) comprises 2588 aligned bp and 21 accessions. ML and Bayesian analyses revealed identical topologies. Axyris is monophyletic with high support and it is recovered as a sister clade to Krascheninnikovia and Ceratocarpus ( Figure 6; BSL 100; PP 1). Axyris amaranthoides is resolved as sister to the rest of the species (BSL 91; PP 1).   The diversification of Axyris started in the late Eocene ca. 25.65 mya (stem age, 95% HPD: 36.3-14.97). The crown age of Axyris dates back to ca. 5.11 mya (95% HPD: 10.91-2.82 mya) which suggests that the genus originated during the early Pliocene (the Zanclean stage).

Biogeographic Analyses and Radiation of Axyris
The ancestral area for the crown node of Axyris could be estimated for the regions A (Southern Siberia, Mongolia, NW China) and B (Tian Shan, Pamir Mountains) (AB: p = 0.55, ABE: p = 0.13, A: p = 0.11; ABD: p = 0.10; Figure 7). It is likely that the ancestral Axyris domain can be linked to the geographic area where four species of the genus Plants 2022, 11, 2873 5 of 22 are present today ( Figure 5; marked with orange). For all other nodes (except node 9, ABE: p = 59, ABCE: p = 30), the likely ancestral domain is also confined to AB areas with varying probabilities (Figure 7, see Legend). Further dispersal of Axyris proceeded towards the Himalayas/Tibet (Region D: A. mira and A. prostrata), which was colonized once, and then Eastern Siberia (Sakha Republic, Region E) (A. sphaerosperma) and the Caucasus, Region C (A. caucasica).
The ancestral area for the crown node of Axyris could be estimated for the regions A (Southern Siberia, Mongolia, NW China) and B (Tian Shan, Pamir Mountains) (AB: p = 0.55, ABE: p = 0.13, A: p = 0.11; ABD: p = 0.10; Figure 7). It is likely that the ancestral Axyris domain can be linked to the geographic area where four species of the genus are present today ( Figure 5; marked with orange). For all other nodes (except node 9, ABE: p = 59, ABCE: p = 30), the likely ancestral domain is also confined to AB areas with varying probabilities ( Figure 7, see Legend). Further dispersal of Axyris proceeded towards the Himalayas/Tibet (Region D: A. mira and A. prostrata), which was colonized once, and then Eastern Siberia (Sakha Republic, Region E) (A. sphaerosperma) and the Caucasus, Region C (A. caucasica).

The Distribution of Axyris in Siberia
Four out of six Axyris species-namely A. amaranthoides, A. hybrida, A. prostrata, and A. sphaerosperma-are native to Siberia [3]. According to the latest examination of the genus in Siberia [16], A. amaranthoides is widespread in Southern Siberia, with a few records in Northern and Eastern Siberia; A. hybrida mostly occurs in Southern Siberia with scattered findings in Eastern Siberia; and A. prostrata is present in Southern Siberia. No distribution map of Axyris sphaerosperma was provided by M. Lomonosova [16], but several findings in Altai and Tyva Republics were reported, as was one finding in the city of Yakutsk (Sakha Republic).
According to our investigations, A. amaranthoides is a common plant in most of Siberia, but its records in the northern and northeastern parts of the region represent recent migrations as a consequence of human activities. In Eastern Siberia, it is more frequently found in settlements and along main transport routes, especially in the Lena River basin. A. hybrida occurs across Southern Siberia, mostly in the Altai and Sayan Mountains but also in the adjacent lowlands. The findings of A. hybrida in Sakha Republic (Eastern Siberia) reported by the authors of refs. [16] and [8] are not confirmed, and these records in fact belong to A. amaranthoides. As stated by M. Lomonosova [16] and confirmed by us, the geographic range of A. prostrata is mostly confined to the Altai and Sayan Mountains, with scattered records in the uplands of Baikalia and Krasnoyarsk Krai.

The Distribution of Axyris in Siberia
Four out of six Axyris species-namely A. amaranthoides, A. hybrida, A. prostrata, and A. sphaerosperma-are native to Siberia [3]. According to the latest examination of the genus in Siberia [16], A. amaranthoides is widespread in Southern Siberia, with a few records in Northern and Eastern Siberia; A. hybrida mostly occurs in Southern Siberia with scattered findings in Eastern Siberia; and A. prostrata is present in Southern Siberia. No distribution map of Axyris sphaerosperma was provided by M. Lomonosova [16], but several findings in Altai and Tyva Republics were reported, as was one finding in the city of Yakutsk (Sakha Republic).
According to our investigations, A. amaranthoides is a common plant in most of Siberia, but its records in the northern and northeastern parts of the region represent recent migrations as a consequence of human activities. In Eastern Siberia, it is more frequently found in settlements and along main transport routes, especially in the Lena River basin. A. hybrida occurs across Southern Siberia, mostly in the Altai and Sayan Mountains but also in the adjacent lowlands. The findings of A. hybrida in Sakha Republic (Eastern Siberia) reported by the authors of refs. [16] and [8] are not confirmed, and these records in fact belong to A. amaranthoides. As stated by M. Lomonosova [16] and confirmed by us, the geographic range of A. prostrata is mostly confined to the Altai and Sayan Mountains, with scattered records in the uplands of Baikalia and Krasnoyarsk Krai.

A Geographical Puzzle Concerning A. sphaerosperma
Out of all Axyris species, only A. sphaerosperma possesses a disjunct range, with two fragments ( Figure 4) reported here for the first time. The first (main) fragment is located Plants 2022, 11, 2873 6 of 22 in the Altai and Sayan Mountains, with extensions to the Tian Shan, Pamir, and North Himalayas [1,3,5]. Here we also add the first but expected records for Mongolia (Mongolian Altai, Bayan-Ulgii prov., Tavan-Bogd Mts., Tsagaan-Gol River basin, 4 August 2001, I. Krasnoborov,A. Shmakov,and D. German 82 (NS0009424); and China (Irenchabirga, 9000 ft, 10 September 1879, A. Regel [LE]; Zaisan expedition [Xinjiang prov., Habahe county], Kobuk River basin, 20 July 1914, V. Sapozhnikov s.n., all as A. amaranthoides [LE]) that have not been mentioned earlier (e.g., [6,17,18]). These new findings were made close to those in Altai Republic (Southern Siberia, Russia). In the main fragment of its range, A. sphaerosperma occurs at high altitudes, between 1500 and 3000 m a.s.l. [19]. The second fragment is located in the lowlands of Eastern Siberia (Sakha Republic). Both areas are characterized by extremely low winter temperatures and a short vegetation period lasting from mid-May to mid-September. The reasons for such a fragmentary range of A. sphaerosperma are unclear. Nonetheless, it should be noted that A. sphaerosperma is well adapted to the harsh climatic conditions by forming a persistent soil seed bank. Although all Axyris species have heteromorphic fruits and seeds with different longevity, some fruits of A. sphaerosperma and A. caucasica have sclereids and contain seeds with a very hard and thick (up to 100-115 µm) seed coat [2]. Such thickness of the seed coat is exceptional for Chenopodiaceae [9,20] and is a good example of physical dormancy in species growing under harsh climatic conditions.

The Origin of Axyris
The origin of this genus is probably connected with the orographic and climatic changes in the late Miocene/early Pliocene, which are manifested in renewed tectonism and further aridification of the lowlands in South Siberia and Tian-Shan [21]. The same region of origin, for example, is reported for Krascheninnikovia ceratoides (L.) Gueldenst. [22,23], a species also belonging to Axyrideae [13]. It is still not clear whether the origin of Axyris and Krascheninnikovia is linked to high-altitude or lowland steppes, but it is thought that the late Miocene and early Pliocene are the time scales where a continent-wide restructuring of the distribution of landscape-forming elements was taking place, including a new zonal structure component: steppe formation [24]. Our time-calibrated tree coincides with previous study [25]. Divergence time between Axyris and Krascheninnikovia + Ceratocarpus group is 26.5 mya and between Krascheninnikovia and Ceratocarpus is 16.4 mya, which generally corresponds with our data (25.6 and 15.9 mya, respectively).
According to the altitudinal gradient, species of Axyris can be subdivided into two main groups: (1) predominantly lowland species (only A. amaranthoides) and (2) predominantly mountain species (all the other taxa), sometimes penetrating onto the lowlands (A. hybrida, A. prostrata, and A. sphaerosperma). None of them can be classified as desert plants, and there is evidently a gap in the distribution of the genus in the Taklamakan desert. A considerable gap between the main distribution area of the genus located in temperate East Asia and a small fragment in the Greater Caucasus (A. caucasica) can be explained by climatic changes, including Paleo-Caspian Transgression in the lowlands of Kazakhstan during the Pliocene and Pleistocene [26]. As stated earlier [1], one type of reproductive diaspores of both A. sphaerosperma and A. caucasica is very thick and provides high seed longevity. The reproductive strategy of these species is connected with the adaptation to extremely low winter temperatures. We suppose that the precursors of these species were present in the Pliocene and early Pleistocene in the lowlands of the Aralo-Caspian floristic province reaching the Caucasus, the areas where permafrost never disappeared during warm phases [27]. Decreasing permafrost led to the desertification of the landscape [28] and therefore could induce the disappearance of cold-adapted species in the lowlands and their isolation in the areas with much colder winter temperatures.

Systematics of Axyris
This topic is elaborated based on a molecular phylogeny for the first time here. As proposed earlier [1,2], carpological data fully support the new systematic subdivision of Axyris into three sections.
Monoecious annuals covered with stellate hairs sometimes intermixed with simple multicellular hairs. Leaves short-or long-petiolate; blades ovate, oblong, spatulate, or lanceolate, entire, rarely undulate. Male flowers arranged in terminal spike-like inflorescences up to 8 cm long, with minute perianths of five free hyaline segments and with 2-5 stamens; female flowers located in bract axils, with five prominent hyaline perianth segments (two of which are erroneously called bracteoles). Fruits always dimorphic (heterocarpous); pericarp tightly adhering to the seed coat, usually with ear-like appendages at the apex of the fruit. Seeds also dimorphic (with thick and thin testal layer of the seed coat). Embryo vertical, horseshoe-shaped (in flattened fruits), or annular (in spheroidal fruits); perisperm present.
Six species in Eurasia, predominantly in Central Asia; one (A. amaranthoides) grows as an alien in many parts of Europe and North America.
One species, Axyris amaranthoides L. (type species of the genus).
Type species: Axyris hybrida L. Three species: Axyris hybrida L., A. prostrata L., and A. mira Sukhor., in Central Asia, Southern Siberia, and the Himalayas/Tibet. Two of them are present in Russia.

Materials and Methods
The taxonomic revision of the herbarium material was conducted at ABGI, G, LE, LECB, MHA, MSK, MSKU, MW, MWG, MOSP, NS, NSK, PE, PVB, RV, RWBG, TK, TLT, VOR, and WIR. The field investigations were carried out by the first author (A.P.S.) in Southern Siberia and the Far East (Amur Oblast). The data on the distribution of A. mira and A. prostrata in Himalaya and Tibet were taken from ref. [1]. All records for each species are given in Appendix A. Distribution maps are based on the specimens cited and were

Sampling and DNA Extraction, Amplification, and Sequencing
Sixty accession numbers were included in the phylogenetic analyses, representing all six Axyris species, as well as 24 accession numbers as outgroups from Chenopodiaceae/Amaranthaceae; the samples are listed in Table 1. DNA was extracted from 5-10 mg of dried leaf samples from the herbarium specimens by means of the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). One nuclear (the nuclear ribosomal internal transcribed spacer, nrITS) and three plastid markers (proteincoding gene rbcL and two intergenic spacers: atpB-rbcL and trnL-trnF) were selected for the phylogenetic analysis.
PCRs were carried out in Thermal Cycler T100 (Bio-Rad, USA) using the primers and cycler programs listed in Table 2. Table 2. Primers and cycler programs used for DNA amplification.

ITS
PCR products were purified with the Cleanup Mini BC023S Kit (Evrogen, Russia). Sanger sequencing was carried out at Evrogen JSC (Moscow, Russia); the sequencing primers were the same as the amplification primers.

Sequence Alignment, Phylogenetic Analyses, and Molecular Dating
Sequences were aligned with MAFFT v.7 at default parameters [35], and the alignment was adjusted manually in PhyDe v.0.9971 [36]. Gaps were treated as missing data during the phylogenetic inference.
Two separate analyses were performed on nuclear and plastid DNA datasets via Bayesian inference (BI) and ML. According to the Akaike information criterion (AIC), the best-fitting model was the GTR + G model for the plastid dataset and the GTR + G + I model for the nuclear dataset, respectively. For the ML analyses, we employed RAxML v.8 [37]. Bootstrap analyses were conducted with 2500 replicates for ML. Due to the lack of statistically significant incongruence between nuclear and plastid trees ( Figures S1 and S2), a combined sequence matrix was compiled for further analysis.
Divergence times for Axyris taxa were estimated using a Bayesian uncorrelated lognormal relaxed clock under a birth-death speciation process [38] for the combined dataset. We selected a normal distribution for the secondary calibration with a mean of 59.2 and standard deviation of 4.3, equivalent to the 95% HPD estimate from ref. [39] for the crown of Chenopodiaceae s.str. Bayesian analyses were conducted in BEAST v.2.6.7 [40]. Four Markov Chain Monte Carlo analyses with four chains were run for 20 million generations for every dataset, with sampling every 20,000 generations. Output log files were analyzed by means of TRACER v.1.6 [41] to assess convergence and ESS of all parameters; 15% of samples were removed as burn-in prior to combining the independent runs with the help of LOGCOMBINER v.2.6.7 [40]. The maximum clade credibility tree was generated using TREEANNOTATOR v. 2.4.5 [40].

Biogeographical Analysis
Geographic distributions of all the studied species were inferred from herbarium specimens. Eight large geographic regions reflecting the worldwide distribution of Chenopodiaceae s. str. were coded as follows: The BI gene trees were pruned to remove all duplicate accessions using the drop.tip function in the ape package [42]. Ancestral range estimation was conducted by means of the time-calibrated tree representing six species of Axyris with only one accession per species using "BioGeoBEARS" [43,44] in R v.4.1.3 [45]. The coded geographic data are displayed in Table 3. We ran the analysis in accordance with a dispersal-extinction-cladogenesis model (DEC model), dispersal-vicariance model (DIVALIKE model), or BAYAREA model (BA-YAREALIKE model) and examined a second run adding parameter "j" (founder-event speciation) for each biogeographic model. Out of the six models explored in this study, the DEC + J model was the best fit judging by the AIC and likelihood ratio test results ( Table 4). The analyses were unconstrained (without possible dispersal routes or ancestral areas assumed a priori). We allowed the inferred ancestor to occupy a maximum of three areas corresponding to the largest number of areas occupied by any extant species.

Conflicts of Interest:
The authors declare no conflict of interest.