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Phylogenetic relationship and biogeography of the genus Diestramima, a cave cricket endemic to the Oriental realm (Rhaphidophoridae: Aemodogryllinae)


 Background

 Diestramima is only distributed in the Oriental realm, and mainly in China. Because they are apterous, these insects are good materials for studying biogeography. In this study, we reconstruct the phylogeny tree of Diestramima species based on maximum likelihood and Bayesian inference based on six genes, and use fossil-based molecular dating and ancestral range estimation to reconstruct the dispersal route.
Results

The results of molecular system are highly consistent with the morphological characteristics of these species. At the subgenus level, it is difficult to give a reasonable explanation on the phylogenetic tree although we found that some groups share some common characteristics. Dating and historical biogeography analyses suggest an early Miocene origin of the genus. Some ancestors of the genus Diestramima have been distributed in Guangxi, Yunnan province and other regions of China at this time. In the late Miocene, the genus Diestramima dispersed from the low mountain and hilly areas of Guizhou, Guizhou and Hunan to inland China. Quaternary glaciation determines their current geographical distribution pattern. Southeast Asia is the most likely original place of the genus Diestramima.
Conclusion

In phylogenetic clades of Diestramima, similarity in geographic distribution is as important as similarity in morphological characteristics. Combined with the results of biogeographic analysis, the two temperature rises in the Palacocene and Miocene may have intensified the dispersal of the ancestors of the genus Diestramima in the Oriental realm. The temperature drop and the uplift of the Qinghai-Tibet Plateau were the main reasons for the divergence within the genus Diestramima. In addition, changes in humidity and changes in surface plant communities are also important factors affecting their divergence.


Introduction
Cave cricket (Rhaphidophoroidea Walker, 1869) as one of the oldest species of Orthoptera, are widely distributed all over the world. It considered to be the basis of the six families and one of the oldest taxa in the Tettigoniidea clade [1,2]. They are gregarious and typical apterous groups, which includes 10 subfamilies and 87 genera (including 1 fossil subfamily and 2 fossil genera). They mainly inhabit in dry leaves, stone crevices, caves and other dark humid environments, therefore their speciation as well as population structure have relatively strong dependence on geographical isolation [3].
Dating estimates indicated that Rhaphidophoridae originated in the Cretaceous period during the Mesozoic era with the ancestral area located both in the northern and southern hemisphere. The current distribution of Rhaphidophoridae might be explained by a combination of both dispersal and vicariance events occurred especially in the ancestral populations [4]. The radiation of Rhaphidophoridae started within the Pangaea, where the ancestor of Rhaphidophoridae occurred throughout an ancestral area including Australia, North America, and the Mediterranean region. The opening of the Atlantic Ocean promoted the divergence of North American and Mediterranean lineages while the differentiation of the southern lineages, spread from Australia, appears to be related to the fragmentation of Gondwana land [4]. The Indian subcontinent (including India and southeast Asian countries north of the Indian river) was originally part of Gondwana continent, and the initial collision between it and Eurasia probably began in the late cretaceous, and the main collision period occurred around 55-50Ma, which coincided with the isolation of the Aemodogryllinae and Rhaphidophorinae. After the collision of the Indian subcontinent and the Eurasian continent, insects from the two continents met again here, which also provided rich raw material for later Oriental insect species [5].
China straddles Palaearctic and Oriental realm, occupies an important biogeographical position in the world. Recent years, many papers on molecular biogeography of insects have been carried out in China. Based on the phylogenetic relation and distribution of four grasshopper genera of Catantopidae from the south of China, ten areas of endemism have been recognized [6]. The order of the formation of the 10 distribution areas of these insects was as follows: southern Yunnan and Loess Plateau, Hainan Island, eastern Mountain and Taiwan Island; The Yunnan-Guizhou plateau, northern Yunnan and Qinling-Daba mountains formed at the same time, while the southern China and jiangnan hilly region formed the last. Wang et al. determined the phylotaxonomic relationship and differentiation time of the genus Sinocyrtaspis endemic to China [7]. Based on the geographic dynamics and climatological data of its ancestors, it was concluded that the centres of origin of Sinocyrtaspis were Hunan and north-east Guangxi. Their ancestors had two dispersal routes ending in Jiangxi and Zhejiang, after the cold period of the Miocene, some of the ancestral species remained in Guizhou while others moved out. And as a response to climate change, species altered distribution areas by moving along altitude gradients in mountain regions, whereas the anthropogenic global warming trend has promoted some species moving to high-latitude areas or caused population differentiation. However, there is still a lack of biogeographic research on the widely distributed Oriental species, especially the Rhaphidophoridae in Oriental realm.
China is the main distribution area of Rhaphidophorinae Walker, 1869 and Aemodogryllinae Jacobson, 1905. Diestramima Storozhenko, 1990 [8] is (Rhaphidophoroidea: Aemodogryllinae, Kevan, 1977) [9,10] widely distributed in the Oriental realm of China (with 70% of known species) and adjacent countries. Their range is limited to the Oriental realm (tropical and subtropical mountainous hilly areas), because they usually choose wide shrub leaves as mating sites. All species of this genus has long protuberances on the seventh abdominal tergite, and the shape of the protuberances is signi cantly different among different species. The morphological classi cation system and the relationship between species are relatively clear. Therefore, Diestramima is a suitable candidate group for studying the speciation and diversity patterns of Oriental realm in China and the corresponding geological changes.
In this paper, we used an integrative approach to identify species, including morphology and multiple methods for molecular identi cation, which clari ed the present taxonomic problems on Diestramima. We estimated the temporal and spatial divergence of this genus, and present a historical framework for the phylogeny and biogeography of these Oriental realm endemic cave crickets. And in the discussion, we try to predict the historical dispersal pattern of pure Oriental species in China from this groups.

Material And Methods
Taxon sampling A total of 87 individuals from 19 species were collected by hand or sweep net at night. Adults were preserved in 70% ethanol in the eld. The morphological identi cations prior to DNA extraction were made depanding on the male apex of abdomen and paraproct [10]. Samples of genus Diestramima were collected from twelve provinces in China Oriental realm, including Anhui, Chongqing, Fujian, Guangdong, Guangxi, Guizhou, Hubei, Hunan, Jiangxi, Sichuan, Yunnan and Zhejiang (Fig. 1). All widespread species in China and a cross-border species distributed in China and Vietnam have been collected. Moreover, we examined a large of additional specimens and relevant literature [9][10][11][12][13][14][15][16][17][18] of Rhaphidophoroidea for better understanding the morphological variations of species, to aid in accurate species determination. Thus, the present sampling is the almost comprehensive one for the phylogenetic analysis in terms of both taxonomy and distribution. All diagnostic characters were photographed by OLYMPUS DP27, and arranged using Adobe Photoshop CC (Fig. 2, Fig. 3).

DNA extraction, ampli cation and sequencing
The total genomic DNA was extracted from the muscles of one hind leg by AxyPrep Genomic DNA Miniprep Kit (AXYGEN), according to the manufacturer's instructions. We used previously published sequences from the family Rhaphidophoroidea to jointly complete this sampling, including six species of the genus Diestrammena Brunner von Wattenwyl, 1888, four species of the genus Rhaphidophora Serville, 1838, twenty species of the subfamily Dolichopodainae Brunner von Wattenwyl, 1888, nineteen species of the subfamily Troglophilinae Krauss, 1879 [4]. The samples and sequence information were listed in Table S1. The following six gene regions were ampli ed: Mitochondrial ribosomal 16S, nuclear ribosomal 28S, mitochondrial cytochrome oxidase I (COI), ATPase catalytic subunit A-like (ACSL), cytoplasmic-like (CPL) and water dikinase-like (WDL). PCR was performed using 2×Es Taq Master Mix (Dye) manufactured by Cowin Bio (China), with a nal volume of 50µL, composed of: 25µL of the 2×Es Taq Master Mix (Dye), 19µL of the ddH2O, 2µL of forward and reverse primer each and 2µL of the sample DNA extract. The PCR annealing temperature for each gene was modi ed (Table S2). PCR products were directly sequenced.

Phylogenetic analyses
We used all Diestramima samples as well as the samples of adjacent genera Diestrammena and Raphidophora in the phylogenetic tree construction. For each fragment, DNA alignment was conducted by MEGA7 software [19,20]. Saturation level of sequences was veri ed by Xia's test [21] in dambe 7 [22]. In PhyloSuite [23], the best-t evolutionary model GTR was derived from ModelFinder [24], and the Maximum likelihood (ML) analysis was performed in IQ-Tree [25] with 1000 ultrafast bootstrap replicates.
Node support is shown as posterior probabilities and bootstrap value, as directly estimated from the majority rule consensus topology.

Divergence time estimation
The phylogenetic of Diestramima using BEAST v. 1.8.4 [26] to reconstruct by selecting the Birth-Death tree prior and the Piecewise linear and constant root of population size model. Given the absence of reliable fossil records for Diestramima, we relied on secondary calibrations based on the available worldwide dated framework of Rhaphidophoroidea [4], we used representatives of four lineages (subfamily Dolichopodainae and Troglophilinae; genus Diestrammena and Rhaphidophora) that were sampled in this study, including 49 cave cricket species. In their conclusion, the ancestor of the Aemodogryllinae and Rhaphidophorinae spread from Australia to south-eastern Asia at 117Ma (95% HPD: 105-130 Ma), an estimate consistent with plate tectonics as India separated from other parts of Gondwana at 130 Ma, prior to other paleogeographic events [27]. Therefore, in our study, it was used as the rst divergent calibration time to prove the purity of these Oriental realm species.
The divergence times of major nodes were estimated under uncorrelated relaxed clock by setting four fossil calibrations: (1) due to the plate tectonics as India separated from other parts of Gondwana, the ancestor of the Aemodogryllinae and Rhaphidophorinae spread from Australia to south-eastern Asia at 117 Ma [4]; (2) the opening of Mid-Aegean trench (9-12 Ma) separated the Troglophilus Krauss, 1879 species inhabiting the eastern Aegean islands and Asia Minor from the Cyclades, Crete and mainland Greece [28]; (3) the uplift of Anatolian Plateau happened 5-10 Ma due to acceleration of northward movement of the Arabian Plate [29] and separating the Anatolian species of Troglophilus from the Balkans ones; (4) the isolation of Crete island, inhabited by Dolichopoda paraskevi Boudou-Saltet, 1973 (Aegean Sea, Greece) from neighboring mainland, happened at the end of Messinian salinity crisis (5.33 Ma) [30]. Trees were sampled every 10000 generations for a total of 100 million generations. We further checked whether the effective sample size (ESS) was greater than 200 for most important parameters using Tracer v.1.7.1. The consensus tree was exported using TreeAnnotator v.1.8.4 [31], discarding the rst 25% of trees as burn-in. The mean node heights and 95% highest probability density (95% HPD) were Ancestral ranges were reconstructed using RASP 4.0 [33] beta. As a guide tree for statistical dispersalextinction-cladogenesis (S-DEC) analyses [34], we removed all outgroups and constructed a phylogenetic tree for the genus Diestramima. Modify and guide this tree according to the maximum credibility tree from the BEAST analysis. Ancestral areas were limited to no more than three areas. Statistical dispersalvicariance (S-DIVA) analysis [35] were performed based on a sample of 1000 random post burn-in RASP trees and use the nal tree. The maximum number of ancestral areas was limited to three.

Species delimitation and phylogeny
Phylogenetic trees (Fig. 4) constructed either by BI or ML analyses had the same topology. Between species, Posterior probability (PP) of all nodes is 1 and bootstrap support values (UFB) are all above 98 except D. (D). eurya is 94. Based on different tree-constructing methods as well as single or combined dataset, the monophyly of the species in Diestramima was well-supported. The membership of each of these major clades were not strongly correlated with the distribution of their subgenera (Baculitettix, Diestramima and Excisotettix) [14]. The species within each clade live in roughly the same area, especially in the southwestern border region of China. D. conica, D. yunnanensis. D. sp. 2, D. major, D. subtilis, D. cycla and D. triangulata are in a clade, although they contain two subgenera. In the southwestern border region of China, even within the same species, samples from different regions showed great variability. We collected D. conica samples from Taiyanghe Reserve, Gaoligongshan and Laifengshan, and the samples from these three regions in Yunnan showed a high divergence. Similarly, D. subtilis samples from three regions (Xiaojie town, Gulinjing town and Ailaoshan) in Yunnan also produced this high divergence. However, widely distributed species in inland areas of China always tend together. We collected D. beybienkoi samples from seven regions (Jinyunshan, Emeishan, Zhangjiajie, Dujiangyan, Leigongshan, Simianshan and Yangmeiao) in ve provinces (Sichuan, Chongqing, Hunan, Guizhou and Guangxi) of China, but they only divergence two clades. This divergence phenomenon is more prominent in D. intermedia and D. austrosinensis. Although the samples we collected spanned several provinces and regions in China, there were basically no divergence within their species.

Divergence time estimation
The divergence time tree is divided into two branches: Aemodogryllinae and Rhaphidophorinae from Asia, Dolichopodainae and Troglophilinae from Europe. We analyze the Asian taxa separately (Fig. 5). The BEAST analysis indicated that the subfamily Aemodogryllinae and Rhaphidophorinae have been diverged since Cretaceous. The speciation events in genus Diestramima occurred from late Miocene to the early Pliocene, and most species was completed in or before early Quaternary. Following the speciation of four Yunnan and Guangxi lineages (D. conica, D. major, D. subtilis and D. yunnanensis) diverged at 8. 32  Ancestral range estimation S-DEC and S-DIVA analyses showed a highly similar biogeographic patterns for Diestramima species and the results shown in Fig. 6. The reconstructed topological tree is highly consistent with the BEAST tree, the lineages located in Guangxi and Yunnan are still the rst to be diverged. The ancestral range for Diestramima in China covers mountainous region of southern Yunnan (A) and mountainous hilly region of Yunnan and Guangxi (B). As for the two clades that followed, the ancestral distribution changed from mountainous region of southern Yunnan (A, point 152) to mountainous region of southern Yunnan and low mountainous hilly region of Guangxi, Guizhou and Hunan (AC, point 145). The ancestral distribution regions of all reconstructed species in both clades include the low mountainous hilly region of Guangxi, Guizhou and Hunan (C). The species in Sichuan basin (D) came from low mountainous hilly region of Guangxi, Guizhou and Hunan (C). Jiangnan hilly region (E) and mountainous hilly region of Fujian and Guangdong (F) greatly promoted the change of the distribution of widespread species in inland China.
The main dispersal routes are indicated by arrows in Fig. 7.

Phylogenetic relationships and systematics
Our phylogenetic results (Fig. 4), based on the combined analysis, are highly similar to the morphological analysis (Fig. 2, Fig. 3). Based on the results from our phylogenetic analyses, species in genus Diestramima that are morphologically similar and geographically area similar tend to be grouped together. Due to the lack of samples from Southeast Asian countries, we compared our data with the descriptions of some species of Diestramima by Gorochov et al. (Fig. 8) [10] and proposed some species that may be closely related. There is currently no description of the male D. yunnanensis. We compared the genital plate of female individuals (Fig. 8. F) collected in Xishuangbanna Tropical Botanical Garden Chinese Academy of Sciences with the description of this site sample by Gorochov et al. (Fig. 8. A46) and found morphological consistency. Therefore, we supplemented the abdominal apex of male D. yunnanensis (Fig. 8. E) based on the results of collection region and phylogenetic tree. D. yunnansis is highly similar to D. propria (Fig. 8. A31, located in Laos), except that the male has a shorter abdomen apex and paraproct. Laos borders Xishuangbanna Dai autonomous prefecture of Yunnan province in China. Similarly, Myanmar borders Baoshan city of Yunnan province in China. The abdomen apex of male D. conica (Fig. 8. B) is highly similar to D. minor (Fig. 8. A36, located in Myanmar), but has completely different shape of paraproct.
In Fig. 2, Fig. 3, Fig. 8 [14]. Paraproct of the subgenus Baculitettix are stick-like (rather long and thin) but with slightly or moderately higher proximal part. In the subgenus Diestramima and Excisotettix, paraproct rather short and usually high (plate-like), with distal part truncate, angular or having hooks and/or teeth (spinules); or paraproct moderately short, almost S-shaped in pro le, with proximal and distal parts more or less equal to each other in height, and with angular or almost spinose apex. The difference is that the female genital plate of the subgenus Excisotettix with rather large posteromedian notch and angular projections around this notch. But in our current phylogenetic tree, we have not found a reasonable explanation for distinguishing these subgenera. In all our samples, we found small differences in the length and curvature of the male abdomen apex even within the same species. But the length of the paraproct, especially the shape always remains the same. Combining with the strong similarities between the male abdomen apex of many species mentioned earlier, perhaps the specialization of paraproct occupies a more dominant position in the morphological taxonomic research of the genus Diestramima.

Time and space frame of Diestramima evolution
The results of biogeographic analysis of all genetic samples (Fig. 5) are basically consistent with the conclusions drawn by Allegrucci and Sbordoni [4]. With the gradual disintegration of Pangea, Rhaphidophoroidea is divided into two clades. The ancestors of Dolichopodainae and Troglophilinae are mainly distributed in Laurasia and the ancestors of Aemodogryllinae and Rhaphidophorinae are mainly distributed in Gondwana. With the Indian subcontinent drift and collision with Eurasia, the separation of the two clades Aemodogryllinae and Rhaphidophorinae were formed gradually.
In the Paleogene period, we found that the separation of genus Diestramima from Diestrammena diverged at 33.90Ma (95% HDP: 18.85-54.36 Ma) and can also be regarded as the separation of tribe Diestramimini and Aemodogryllini. During this epoch, one of the most signi cant climate changes on Earth since the Cenozoic era occurred: the Eocene/ Oligocene boundary cooling event. It is characterized by the rapid change of the Earth's climate from the previous Greenhouse to Icehouse, and the emergence of Ice sheets in Antarctica is accompanied by a signi cant drop in global temperature [36]. A drop in global temperatures may eventually lead to the divergence of Aemodogryllini, which is more resilient to cold conditions and can be dispersed to a wider area. The extreme temperature rise of the Paleocene-Eocene thermal maximum did not affect the diverged of the Aemodogryllinae and Rhaphidophorinae ancestors, and may have only intensi ed their dispersal in the Oriental realm.
In the Neogene period, the rst major separation time of the genus Diestramima diverged at 8.32Ma (95% HDP: 4.84-12.88 Ma). At this time, temperatures began to warm during the Miocene, and various tectonic processes occurred due to the collision and compression of continental plate drift and the intensi cation of mountain building. Among them, the Himalayan orogeny had the most profound impact on China [37]. It transformed the planetary wind system in mainland China from early Miocene to monsoon wind system; the arid inland areas in northwest China and the monsoon areas in east China began to form. Until the late Miocene, the climate pattern of humid southeast and arid northwest was basically formed. Paleoclimate changes peaked during the Miocene and gradually shaped the modern climate patterns that prevail today [38]. The humid climate in the eastern area and the sea level caused by warming temperatures may cause some species in Diestramima to gradually migrate east and north, and eventually become isolated due to the frequent mountain building activities. In Fig. 5, the differentiation in Diestramima has a certain lag in this gradually humid and warm climate, a reasonable explanation is that their mating behavior is dependent on broadleaf shrubs or dwarf trees. Due to the monsoon climate change, the relationship between the plant communities and vegetation types in Eastern China during this period is also undergoing changes [39][40][41][42]. We found that in the males of the genus Diestramima, they will stay in place after the ejaculation is over and continue to lift and press the female genitalia, and the frequency and method of this behavior vary according to the species. We believe that this behavior may speed up the sperm entry into the female body while also preventing or slowing down the rate at which the female eats the spermatophore. Correspondingly, all species in Diestramima we have observed will choose dwarf trees, shrubs and ferns with large leaves as their venues for this mating behavior. This behavior has not been found in other species in the family Aemodogryllinae. austrosinensis, D. beybienkoi, D. intermedia) in inland areas of China diverged almost at the same time (2.54-1.02 Ma). The temperature drop and ice sheet increase brought about by the Quaternary glaciation also affected their spread at this time. The distribution and ancestral proliferation characteristics of the genus Diestramima in China are very similar to the ancestral distribution characteristics of the genus Sinocyrtaspis [7] and the four genera of Catantopidae [6] in China. The mountainous region of southern Yunnan is one of the important origins of these grasshopper genera of Catantopidae ancestors. The centres of origin of Sinocyrtaspis were Hunan and north-east Guangxi, and they spread with this place as the center. This may be one of the dispersal paths of Orthoptera in China.

Conclusions
Here we according to the morphological characteristics and the phylogenetic tree constructed based on the 6 genes, phylogenetic relationship of the genus Diestramima is shown. At present, the interspecies morphological characteristics and molecular phylogeny relationships of all the samples have maintained extremely high consistency. However, at the subgenus level, it is di cult to give a reasonable explanation on the phylogenetic tree although we found that some groups share some common characteristics. In phylogenetic clades of Diestramima, similarity in geographic distribution is as important as similarity in morphological characteristics. This phenomenon may be related to their lack of wings and weak mobility. Combined with the results of biogeographic analysis, the ancestors of the genus Diestramima had already arrived in Guangxi and Yunnan provinces in China before the Miocene. The two temperature rises in the Palacocene and Miocene may have intensi ed the dispersal of the ancestors of the genus Diestramima in the Oriental realm. The temperature drop and the uplift of the Qinghai-Tibet Plateau were the main reasons for the divergence within the genus Diestramima. In addition, changes in humidity and changes in surface plant communities are also important factors affecting their divergence. The low mountainous hilly region of Guangxi, Guizhou and Hunan is the area where the insects of this genus are most differentiated. At the early Pliocene, they may have reached most of the areas where they are now.
The arrival of the Quaternary Ice Age has caused these species to undergo violent and complex evolution in inland China.

Declarations
Availability of supporting data The data sets supporting the results of this article (Additional le 1: Tables S1 and S2) are available as online Additional le 1.

Competing interests
The authors declare no competing interests.
Authors' contributions JSZ, YYQ and CZS collected the data, led the writing and conceived the idea, and analyzed the data; ZQH and KL led the writing. All authors read and approved the nal manuscript.     Historical biogeography of Diestramima as inferred by ancestral area reconstructions carried out with RASP under S-DEC and S-DIVA model. Showing estimations in color charts. The number represents the optimal distributions at each node.

Figure 7
The map represents the six major biogeographical areas and dispersal routes of Diestramima: mountainous region of southern Yunnan (A), mountainous hilly region of Yunnan and Guangxi (B), low mountainous hilly region of Guangxi, Guizhou and Hunan (C), Sichuan basin (D), Jiangnan hilly region (E) and mountainous hilly region of Fujian and Guangdong (F).