Solenysa, a Cretaceous Relict Spider Group in East Asia

Abstract

A time scale of phylogenetic relationships contributes to a better understanding of the evolutionary history of organisms. Herein, we investigate the temporal divergence pattern that gave rise to the poor species diversity of the spider genus Solenysa in contrast with the other six major clades within linyphiids. We reconstructed a dated phylogeny of linyphiids based on multi-locus sequence data. We found that Solenysa diverged from other linyphiids early in the Cretaceous (79.29 mya), while its further diversification has been delayed until the middle Oligocene (28.62 mya). Its diversification trend is different from all of the other major lineages of linyphiids but is closely related with the Cenozoic ecosystem transition caused by global climate changes. Our results suggest that Solenysa is a Cretaceous relict group, which survived the mass extinction around the K-T boundary. Its low species diversity, extremely asymmetric with its sister group, is largely an evolutionary legacy of such a relict history, a long-time lag in its early evolutionary history that delayed its diversification. The limited distribution of Solenysa species might be related to their extreme dependence on highly humid environments.

1. Introduction

Molecular data has become an indispensable tool for the reconstruction of phylogenetic relationships among species and provides important insights on the evolutionary histories of many animal groups. It is common in systematics that a high-level molecular phylogeny may significantly conflict with the established taxonomic system based on morphological characters [1,2,3,4]. Understanding the evolutionary past that shaped the species diversity of lineages always attracts the interest of biologists [5,6,7,8]. Spiders are generalist predators, forming a successful terrestrial animal group, and their high species diversity is distributed unevenly across lineages [9], even extremely asymmetrically between sister groups [4,10]. Several hypotheses have been proposed to interpret the driving forces that promote spider diversification, such as co-diversification with insects [11,12,13], key innovations in silk structure and web architecture [10], repeated evolution of the respiratory system from book lungs to tracheae [14], and foraging changes from using capturing web to cursorial habits [15]. While these studies usually focus on the driving forces for fast diversifications that lead to a speciose clade, little attention having been exerted to the factors that might result in groups with poor species diversity. Herein, we investigated the evolutionary history of Solenysa spiders, one of the seven main clades within linyphiids, with poor species diversity compared to other clades [4].

Great conflicts exist between the molecular phylogeny of linyphiids and the classical taxonomic system. Linyphiidae is an ancient spider group and its earliest fossil record dates back to the early Cretaceous, about 125–135 mya [16]. As with many other spiders, linyphiids have experienced adaptive radiation, accompanying the fast radiation of insects in the Cretaceous. Currently, with more than 4700 recognized species, Linyphiidae represents the second largest group of the order Araneae [17]. Generally, linyphiids are conservative in somatic features but have complex genitalia with species-specific characters, which are used as criteria for species recognition. The classical taxonomic system of Linyphiidae consists of seven subfamilies [18]. However, this was not supported by molecular phylogenetic analyses [1,4,7]. Four of them, Linyphiinae, Erigoninae, Micronetinae, and Ipainae, were not monophyletic groups; the representatives of Mynogleninae and Dubiaraneinae fell into Linyphiinae; the Stemonyphantinae taxa were often clustered with pimoids, the sister group of linyphiids [1,4,19,20,21]. The subfamily Stemonyphantinae was newly revised by adding two ex-pimoid genera and another linyphiid genus in it [8]. However, the seven-clade topology of molecular phylogenies are robustly supported, and all of these seven major clades (clades A–F and S in [4]) are supported by some putative synapomorphic characters.

Spider diversity is distributed unevenly among the seven major clades within linyphiids, especially between sister groups [4]. The relationships among the seven major lineages are puzzling. The cladogenetic events of the seven-clade topology were correlated with successive transformations on the state of the epigynal plate that was defined by the location of the copulatory openings and tracings of epigynal tracts [4]. Generally, a set of epigynal characters forms an epigynal type that means a certain interaction pattern between male and female genitalia during copulation [22]. The series of state transformations of the epigynal plate coupled with those lineage divergence events make the seven-clade topology meaningful [4]. Nevertheless, the driving forces that shape the unbalanced diversification across lineages within linyphiids remain unresolved. Among them, the extremely asymmetric species diversity between Solenysa (15 spp.) and its sister group (2885 spp., [18]) provides us a model system to study the evolution of such an asymmetry.

Clade S in [4] is composed of a single genus, Solenysa, and is a unique lineage in linyphiids. All Solenysa species display a distinctive somatic appearance and special genital morphology. These make them easily distinguished from all other linyphiids [23,24]. However, the placement of Solenysa within linyphiids has long been controversial. Saaristo [25] placed Solenysa together with some micronetine genera into a new subfamily, Ipainae, largely based on the females having a movable epigynum. However, this treatment was disproved by the phylogenetic analyses either based on molecular data or morphological data [1,4,24]; the molecular phylogeny supported a sister relationship between Solenysa and clade B, which is a hodgepodge composed of all erigonines, some micronetines, and linyphiines. Although such a sister-relationship was supported by some putative synapomorphies, in comparison to its speciose and widespread sister-clade, the Solenysa clade appeared unusual in term of species diversity and limited distributions and is generally only known from type localities and small adjacent areas (Figure 1; [23,24,26,27,28,29,30,31,32]). The underlying evolutionary process that gave rise to such a biased diversity pattern between these sister clades remains to be explored.

Figure 1. Distribution of Solenysa species. Circles in color represent species of different clades in Figure 2, green, clade L; blue, clade W; purple, clade J, red, clade P. Dash line circles indicate locations of some supposed Pleistocene ice age refugia in East Asia.

Studies on the defining features (synapomorphies) of a speciose group, especially in comparison to its sister group, may help us in search of potential drivers that promote fast diversification [9]. According to Wang et al. [4], the tracheal system in clade B repeatedly evolved from the haplotracheate to desmitracheate, in which the median pair tracheae extensively branched and extended into the prosoma [33]. Especially the distal erigonines clade, all species have a desmitracheate system forming the largest group in clade B [34], contributing more than half of the species diversity of Linyphiidae. While the tracheal system in Solenysa remains as an intermediate type, with the median pair unbranched in the opisthosoma but extended into the prosoma where they branch and extend into the legs (Tu, personal observation; [24]). Anterior extending tracheae would provide oxygen directly to the brain and legs [35,36], and extensively branched tracheae may help in reducing water loss [9,37]. These imply that the selective advantages of the desmitracheate system might trigger the fast diversification in clade B, while the diversification in the Solenysa clade remained slower, which resulted in the biased species diversity between them. Nevertheless, whether there are any other reasons is unclear.

In the present study, we aimed to explore the reason that caused the biased diversification between Solenysa and its sister group. Our results from phylogenetic analyses, molecular dating, and lineage-wise diversification tracing through time suggested that Solenysa represents a Cretaceous relict spider group in East Asia, and historical climate changes have played a pivotal role in shaping its evolutionary history.

2. Materials and Methods

2.1. Taxon Sampling

In the present study, we collected and sequenced four additional Solenysa species, S. lanyuensis, S. yangmingshana, S. macrodonta, and S. ogatai from their type localities on Taiwan Island and Japanese islands. The relevant sequence data for nine Solenysa species, S. longqiensis, S. retractilis, S. tianmushana, S. wulingensis, S. protrudens, S. mellotteei, S. trunciformis, S. reflexilis, and S. partibilis, have been generated in our earlier study [4]. Another two known species, S. spiralis from Sichuan, China, and S. geumoensis from the Korean Peninsula, were not sampled due to a lack of fresh materials for sequencing. Thus, 13 of the 15 known Solenysa species (86.7%) were included in the present study. To explore the phylogenetic position of Solenysa species within Linyphiidae, other linyphiid taxa of Wang et al. [4], except for the four unstable long-branch taxa and those repeated taxa, were compiled in our dataset. Pimoidae is the sister group of Linyphiidae and often used as outgroups for rooting. Given the recent revision of Pimoidae having the formerly pimoid genera Putaoa and Weintrauboa transferred to Linyphiidae, and redelimited Pimoidae as including only Pimoa and Nanoa [8], we also added representatives of Pimoa and Putaoa into the dataset. The final data set consisted of 127 taxa, including 13 Solenysa taxa, 113 other linyphiids, and one pimoid.

Collected information on the four newly sequenced Solenysa specimens is listed in Supporting Material Table S1. Specimens used for the molecular study were fixed in 95% ethanol and kept at −20 °C before DNA extraction. All newly collected specimens are deposited at the College of Life Sciences, Capital Normal University (CNU).

2.2. Laboratory Protocols for Molecular Data

Five loci used in [4], including two mitochondrial genes, cytochrome c oxidase subunit I (COI), and 16S rRNA (16S), and three nuclear genes, 18S rRNA (18S), 28S rRNA (28S), and histone H3 (H3), were sequenced for newly collected Solenysa specimens. Laboratory protocols and sequence curation follow those described in Wang et al. [4]. The primers and their annealing temperatures used for PCR amplification in the present study are provided in Table 1.

Table 1. Primer sequences, their sources, and reaction conditions used for PCR in this study.

Gene	Primer	Sequence (5′–3′)	Annealing Temperature	Voucher
COI	C1-J-1718(F)	GGA GGA TTT GGA AAT TGA TTA GTT CC	45 °C	Simon et al., 1994
	C1-N-219(R)	CCC GGT AAA ATT AAA ATA TAA ACT TC		Simon et al., 1994
16S	LR-N-13398 (F)	CGC CTG TTT AAC AAA AAC AT	42–45 °C	Arnedo et al., 2004
	LR-J-12864 (R)	CTC CGG TTT GAA CTC AGA TCA		Simon et al., 1994
18S	18Sa2.0 (F)	ATG GTT GCA AAG CTG AAA C	58 °C	Giribet et al., 1999
	5F (F)	GCG AAA GCA TTT GCC AAG AA		Giribet et al., 1999
	9R (R)	GAT CCT TCC GCA GGT TCA CCT AC		Giribet et al., 1999
28S	28Sa (F)	GAC CCG TCT TGA AAC ACG GA	48 °C	Whiting et al., 1997
	28Sb (R)	TCG GAA GGA ACC AGC TAC TA		Whiting et al., 1997
H3	H3a (F)	ATG GCT CGT ACC AAG CAG AC(ACG) GC	48 °C	Colgan et al., 1998
	H3b (R)	ATA TCC TT(AG) GGC AT(AG) AT(AG) GTG AC		Colgan et al., 1998

2.3. Phylogenetic Analyses

Sequences for the five genes were aligned using MAFFT 7.490 [38] and were concatenated using Mesquite version 3.31 [39] in the order of 16S, 18S, 28S, COI, and H3. Maximum likelihood (ML) phylogenetic analysis was carried out using IQ-TREE v2.1.3 [40] with best-fit DNA substitution models selected using ModelFinder [41]. ML trees were inferred from (1) the concatenated super-matrix with a single overall the best-fit model and (2) the partitioned matrix defined by loci using PartitionFinder [42] with each partition applying its best-fit model. The GTR + F + R5 was selected as the best-fit model for the concatenated super-matrix. The models selected for partitions of the best scheme were: TIM2 + F + I + G4 for partition 16S, SYM + R4 for partition 18S + 28S, GTR + F + I + G4 for both partition COI and partition H3. Node supports were assessed through the ultrafast bootstrap method [43] with 1000 replicates, incurring the -bnni option to reduce the risk of overestimating branch supports. We also performed Bayesian phylogenetic inference based on the best partition scheme using MrBayes v3.2.6 [44]. We used the GTR model for the 16S data in place of the TIM2 model, as the latter is not implemented in MrBayes. The SYM model for the 18S + 28S data was converted from the GTR model by fixing the stationary state frequencies to be equal. MrBayes analyses were initiated with random starting trees employing four Markov chains (one cold and three hot). The Markov chains ran for 2 × 10⁶ generations with trees and parameters being sampled every 100 generations. The “temperature” parameter was set to 0.2. The chains were converged and reached a stationary state after the iteration with the average standard deviation of split frequencies being smaller than 0.0074, and all values of potential scale reduction factor for all parameters being very close to 1.00. The majority-rule consensus tree was generated using the sample from the cold chain after the first 25% of the sample was discarded as burn-in. The topology of phylogeny inferred from the present study was statistically tested for robustness against alternative topologies with the approximately unbiased (AU) test [45].

2.4. Estimation of Divergence Times

The divergence times were estimated with a relaxed molecular clock approach implemented in BEAST2 version 2.6.6 [46]. The rate change was explicitly modeled using uncorrelated lognormal distribution across trees and a birth–death model was used for modeling speciation. The best-fit DNA substitution model was selected using bModelTest module [47] in BEAUti2. Two independent MCMC searches were run for 8 × 10⁷ generations with trees and parameters being sampled every 1000 generations. The convergence of the MCMC chains was checked with Tracer version 1.7.1 [48]. The first 10% samples were discarded as burn-in.

Two calibration points were used: (1) the oldest linyphiid fossil from the Lower Cretaceous Lebanese amber and (2) the divergence between two endemic Hawaiian species Orsonwelles polites and Orsonwelles malus. The oldest linyphiid fossil was originally described as an undetermined linyphiid [16]. Several studies have used it as a calibration point based on different assumptions: as a stem linyphiid, a crown linyphiid, a crown clade containing all linyphiids except Stemonyphantes [7,8,19,20,49,50], or even as a stem araneoid [51]. Herein, following Arnedo and Hormiga [7], we assigned the age of the fossil to the most recent common ancestor (MRCA) of Linyphiidae and applied an exponential prior with a mean of 10.0 and offset = 125.0 for this calibration, which gave a 95% confident interval of 125–155 Myr. According to Hormiga et al. [52], the Hawaiian spiders Orsonwelles malus is endemic to Kaui Island (formed 5.1 million years ago), and Orsonwelles polites is endemic to the adjacent O’ahu Island (formed 2.6–3.7 million years ago). We assigned a normal prior for the divergence time between these two species with a mean of 3.0 Myr and standard deviation of 0.5 Myr, following Arnedo and Hormiga [7].

2.5. Lineages Tracing through Time

We use lineages through time (LTT) plot to gain insight into the history of diversification for clades S (Solenysa), A, and B. Samples of dated genealogies for each of these three clades were inferred using BEAST2. The times of the MRCA for the relevant clades came from the dated phylogeny. The LTT plots were generated using Tracer version 1.7.1 [48].

3. Results

3.1. DNA Sequence Data

A total of 590 sequences were obtained. Sequences for all five genes were acquired for 88 taxa (68.22%; 88/129), and at least four genes were acquired for the majority (96.06%; 122/127). Fragments from 16S, 18S, 28S, COI, and H3 were sequenced for the taxa sampled here are 91.34% (116/127), 97.64% (124/127), 81.89% (104/127), 95.28% (121/127), and 98.43% (125/127), respectively. After alignment, the concatenated matrix includes a total of 2678 sites. All newly acquired sequences have been deposited in GenBank. The accession numbers of all samples are listed in Table 2.

Table 2. Taxon list and sequence information. An asterisk (*) following species names indicate that they are newly sequenced for the present study.

Family	Subfamily	Species	16s	18s	28s	COI	H3	Voucher
Pimoidae		Pimoa sp. X131	AY230940	AY230893	AY231072	AY231025	AY230985	Arnedo et al., 2004
Linyphiidae	Micronetinae	Acanoides beijingensis	KJ027589	KJ027587	KJ027580	KJ027582	KJ027583	Wang et al., 2015
Linyphiidae	Micronetinae	Acanoides hengshanensis	KJ027585	KJ027588	KJ027584	KJ027586	KJ027581	Wang et al., 2015
Linyphiidae	Micronetinae	Agyneta ramosa	FJ838670	FJ838694		FJ838648	FJ838740	Arnedo et al., 2009
Linyphiidae	Micronetinae	Agyneta sp.1	KT003097	KT002904	KT003003	KT002707	KT002804	Wang et al., 2015
Linyphiidae	Micronetinae	Agyneta sp.2	KT003098	KT002905	KT003004	KT002708	KT002805	Wang et al., 2015
Linyphiidae	Micronetinae	Allomengea sp.	KT003099	KT002906		KT002709	KT002805	Wang et al., 2015
Linyphiidae	Micronetinae	Anguliphantes sp.		KT002907	KT003005	KT002710	KT002807	Wang et al., 2015
Linyphiidae	Micronetinae	Anguliphantes zygius	KT003100	KT002908	KT003006	KT002711	KT002808	Wang et al., 2015
Linyphiidae	Micronetinae	Arcuphantes arcuatulus			KT003007		KT002809	Wang et al., 2015
Linyphiidae	Erigoninae	Asthenargus edentulus	KT003101	KT002909	KT003008	KT002712	KT002810	Wang et al., 2015
Linyphiidae	Linyphiinae	Australolinyphia remota	FJ838671	FJ838695	FJ838718	FJ838649	FJ838741	Arnedo et al., 2009
Linyphiidae	Linyphiinae	Bathyphantes eumenis	KT003101	KT002910	KT003009	KT002713	KT002811	Wang et al., 2015
Linyphiidae	Linyphiinae	Bathyphantes gracilis	FJ838672	FJ838696		FJ838650	FJ838742	Arnedo et al., 2009
Linyphiidae	Linyphiinae	Bathyphantes tongluensis	KT003104	KT002912	KT003011	KT002715	KT002813	Wang et al., 2015
Linyphiidae	Micronetinae	Bifurcia curvata	KT003105	KT002913	KT003012	KT002716	KT002814	Wang et al., 2015
Linyphiidae	Micronetinae	Bifurcia ramosa	KT003106	KT002914	KT003013	KT002717	KT002815	Wang et al., 2015
Linyphiidae	Micronetinae	Bolyphantes alticeps	AY078660	AY078667	AY078678	AY078691	AY078700	Hormiga et al., 2003
Linyphiidae	Micronetinae	Bolyphantes sp.	KT003107	KT002915		KT002718	KT002816	Wang et al., 2015
Linyphiidae	Micronetinae	Centromerus trilobus	KT003108	KT002916	KT003014	KT002718	KT002817	Wang et al., 2015
Linyphiidae	Erigoninae	Ceratinella sp.	KT003109	KT002917	KT003015		KT002818	Wang et al., 2015
Linyphiidae	Erigoninae	Collinsia inerrans	KT003110	KT002918	KT003016	KT002720	KT002819	Wang et al., 2015
Linyphiidae	Erigoninae	Concavocephalus rubens	KT002919	KT003017		KT002721	KT002820	Wang et al., 2015
Linyphiidae	Micronetinae	Crispiphantes sp.	KT003111	KT002920	KT003018	KT002722	KT002821	Wang et al., 2015
Linyphiidae	Micronetinae	Denisiphantes sp.	KT003112	KT002921	KT003019	KT002723	KT002822	Wang et al., 2015
Linyphiidae	Erigoninae	Dicymbium libidinosum	KT003113	KT002922	KT003020	KT002724	KT002823	Wang et al., 2015
Linyphiidae	Erigoninae	Dicymbium tibiale	KT003114	KT002923	KT003021	KT002725	KT002824	Wang et al., 2015
Linyphiidae	Linyphiinae	Diplostyla concolor	FJ838673	FJ838697		FJ838651	FJ838743	Arnedo et al., 2009
Linyphiidae	Micronetinae	Doenitzius pruvus	KT003116	KT002925	KT003023	KT002727	KT002826	Wang et al., 2015
Linyphiidae	Micronetinae	Drapetisca socialis	FJ838674	FJ838698		FJ838652	FJ838744	Arnedo et al., 2009
Linyphiidae	Dubiaraneinae	Dubiaranea distincta	FJ838675	FJ838699	FJ838722	FJ838653	FJ838745	Arnedo et al., 2009
Linyphiidae	Micronetinae	Eldonia sp.	KT003117	KT002926	KT003024	KT002728	KT002827	Wang et al., 2015
Linyphiidae	Erigoninae	Erigone prominens		KT002927	KT003025	KT002729	KT002828	Wang et al., 2015
Linyphiidae	Micronetinae	Floronia bucculenta	FJ838676	FJ838700		FJ838654	FJ838746	Arnedo et al., 2009
Linyphiidae	Linyphiinae	Frontinella communis	FJ838677	FJ838701	FJ838724	FJ838655	FJ838747	Arnedo et al., 2009
Linyphiidae	Erigoninae	Gnathonarium taczanowskii	KT003119	KT002929	KT003027	KT002730	KT002830	Wang et al., 2015
Linyphiidae	Erigoninae	Gonatium rubellum	FJ838679	FJ838703		FJ838656	FJ838749	Arnedo et al., 2009
Linyphiidae	Erigoninae	Gonatium rubens	KT003120	KT002930	KT003028	KT002732	KT002831	Wang et al., 2015
Linyphiidae	Erigoninae	Gongylidiellum vivum	FJ838678	FJ838702	FJ838725		FJ838748	Arnedo et al., 2009
Linyphiidae	Erigoninae	Gongylidioides rimatus	KT003121	KT002931	KT003029	KT002733	KT002832	Wang et al., 2015
Linyphiidae	Mynogleninae	Haplinis diloris	FJ838680	FJ838704		FJ838657	FJ838750	Arnedo et al., 2009
Linyphiidae	Micronetinae	Helophora insignis	FJ838681	FJ838705		FJ838658	FJ838751	Arnedo et al., 2009
Linyphiidae	Erigoninae	Hilaira herniosa	KT003123	KT002933	KT003030	KT002735	KT002834	Wang et al., 2015
Linyphiidae	Erigoninae	Hylyphantes graminicola	KT003124	KT002934	KT003031	KT002736	KT002835	Wang et al., 2015
Linyphiidae	Erigoninae	Hypselistes jacksoni		KT002935	KT003032	KT002737	KT002836	Wang et al., 2015
Linyphiidae	Micronetinae	Incestophantes kochiellus	KT003125	KT002936	KT003033	KT002738	KT002836	Wang et al., 2015
Linyphiidae	Linyphiinae	Kaestneria pullata	KT003126	KT002937	KT003034	KT002739	KT002838	Wang et al., 2015
Linyphiidae	Erigoninae	Lasiargus sp.	KT003127	KT002938	KT003035	KT002740	KT002839	Wang et al., 2015
Linyphiidae	Micronetinae	Lepthyphantes hamifer	KT003128	KT002939	KT003036	KT002741	KT002840	Wang et al., 2015
Linyphiidae	Micronetinae	Lepthyphantes luteipes	KT003129	KT002940		KT002742	KT002841	Wang et al., 2015
Linyphiidae	Micronetinae	Lepthyphantes minutus	AY078663	AY078673	AY078681	AY078689	AY078705	Hormiga et al., 2003
Linyphiidae	Micronetinae	Lidia sp.	KT003130	KT002941	KT003037		KT002841	Wang et al., 2015
Linyphiidae	Linyphiinae	Linyphia triangularis	AY078664	AY078668	AY078682	AY078693	AY078702	Hormiga et al., 2003
Linyphiidae	Linyphiinae	Laetesia sp.	FJ838682	FJ838706		FJ838659	FJ838752	Arnedo et al., 2009
Linyphiidae	Micronetinae	Macrargus rufus	KT003133	KT002944	KT003040	KT002745	KT002845	Wang et al., 2015
Linyphiidae	Erigoninae	Maso sundevalli		KT002945	KT003041	KT002746	KT002846	Wang et al., 2015
Linyphiidae	Micronetinae	Meioneta rurestris	FJ838683	FJ838707		FJ838660	FJ838753	Arnedo et al., 2009
Linyphiidae	Micronetinae	Mesasigone mira	KT003134	KT002946		KT002746	KT002847	Wang et al., 2015
Linyphiidae	Erigoninae	Micrargus herbigradus	KT003135	KT002947	KT003042	KT002748	KT002848	Wang et al., 2015
Linyphiidae	Linyphiinae	Microlinyphia dana	AY078665	AY078677	AY078683	AY078690		Hormiga et al., 2003
Linyphiidae	Linyphiinae	Microlinyphia sp.	KT003136	KT002948	KT003043	KT002749	KT002849	Wang et al., 2015
Linyphiidae	Micronetinae	Microneta viaria	FJ838684	FJ838708		FJ838661	FJ838754	Arnedo et al., 2009
Linyphiidae	Micronetinae	Micronetine sp.1	KT003138	KT002950		KT002751	KT002851	Wang et al., 2015
Linyphiidae	Micronetinae	Micronetine sp.2	KT003139	KT002951	KT003045	KT002752	KT002851	Wang et al., 2015
Linyphiidae	Micronetinae	Micronetine sp.3	KT003140	KT002952	KT003046	KT002753	KT002853	Wang et al., 2015
Linyphiidae	Micronetinae	Mughiphantes nigromaculatus	KT003187	KT003001	KT003095	KT002802	KT002902	Wang et al., 2015
Linyphiidae	Micronetinae	Mughiphantes sp.1A	KT003141	KT002953	KT003047	KT002754	KT002854	Wang et al., 2015
Linyphiidae	Micronetinae	Mughiphantes sp.1B	KT003142	KT002954	KT003048	KT002755	KT002855	Wang et al., 2015
Linyphiidae	Micronetinae	Mughiphantes sp.2	KT003143	KT002955		KT002756	KT002855	Wang et al., 2015
Linyphiidae	Micronetinae	Mughiphantes sp.3	KT003144	KT002956	KT003049	KT002757	KT002857	Wang et al., 2015
Linyphiidae	Linyphiinae	Neriene emphana	KT003145	KT002957	KT003050	KT002758	KT002858	Wang et al., 2015
Linyphiidae	Linyphiinae	Neriene radiata	AY078710	AY078670	AY078684	AY078696	AY078709	Hormiga et al., 2003
Linyphiidae	Linyphiinae	Neriene sp.2	KT003146	KT002958	KT003051	KT002759	KT002859	Wang et al., 2015
Linyphiidae	Linyphiinae	Neriene sp.3	KT003147	KT002959	KT003052	KT002760	KT002859	Wang et al., 2015
Linyphiidae	Linyphiinae	Neriene sp.4	KT003148	KT002960	KT003053	KT002761	KT002861	Wang et al., 2015
Linyphiidae	Linyphiinae	Neriene sp.5	KT003149	KT002961	KT003054	KT002762	KT002862	Wang et al., 2015
Linyphiidae	Linyphiinae	Neriene sp.6	KT003150	KT002962	KT003055	KT002763	KT002863	Wang et al., 2015
Linyphiidae	Linyphiinae	Neriene variabilis	AY078711	AY078669	AY078685	AY078699	AY078706	Hormiga et al., 2003
Linyphiidae	Micronetinae	Nippononeta coreana	KT003151	KT002963	KT003056	KT002764	KT002864	Wang et al., 2015
Linyphiidae	Mynogleninae	Novafroneta vulgaris		FJ838710		FJ838663	FJ838756	Arnedo et al., 2009
Linyphiidae	Erigoninae	Oedothorax apicatus	KT003152	KT002964	KT003057	KT002765	KT002864	Wang et al., 2015
Linyphiidae	Erigoninae	Oia imadatei	KT003153	KT002965	KT003058	KT002765	KT002866	Wang et al., 2015
Linyphiidae	Erigoninae	Oreoneta tienshangensis	KT003154	KT002966	KT003059	KT002765	KT002867	Wang et al., 2015
Linyphiidae	Linyphiinae	Orsonwelles malus	AY078737	AY078676	AY078687	AY078697	AY078708	Hormiga et al., 2003
Linyphiidae	Linyphiinae	Orsonwelles polites	AY078725	AY078671	AY078686	AY078755	AY078701	Hormiga et al., 2003
Linyphiidae	Micronetinae	Oryphantes sp.1	KT003155	KT002967	KT003060	KT002768	KT002868	Wang et al., 2015
Linyphiidae	Micronetinae	Oryphantes sp.2A	KT003156	KT002968	KT003061	KT002769	KT002869	Wang et al., 2015
Linyphiidae	Micronetinae	Oryphantes sp.2B	KT003157	KT002969	KT003062	KT002801	KT002870	Wang et al., 2015
Linyphiidae	Erigoninae	Ostearius melanopygius	FJ838688	FJ838712			FJ838758	Arnedo et al., 2009
Linyphiidae	Linyphiinae	Pacifiphantes zakharovi	KT003159	KT002971	KT003064	KT002771	KT002872	Wang et al., 2015
Linyphiidae	Micronetinae	Parawubanoides unicornis	KT003160	KT002972	KT003065	KT002772	KT002873	Wang et al., 2015
Linyphiidae	Micronetinae	Poeciloneta variegata	KT003161	KT002973	KT003066	KT002772	KT002874	Wang et al., 2015
Linyphiidae	Linyphiinae	Porrhomma longjiangense	KT003162	KT002974	KT003067	KT002774	KT002875	Wang et al., 2015
Linyphiidae	Mynogleninae	Pseudafroneta incerta	FJ838690	FJ838714	FJ838737	FJ838666	FJ838760	Arnedo et al., 2009
Linyphiidae	Stemonyphantinae	Putaoa huaping	KT003163	KT002975	KT003068	KT002775	KT002876	Wang et al., 2015
Linyphiidae	Micronetinae	Ryojius sp.			KT003069	KT002776	KT002877	Wang et al., 2015
Linyphiidae	Micronetinae	Saaristoa ebinoensis	KT003164	KT002976	KT003070	KT002777	KT002878	Wang et al., 2015
Linyphiidae	Erigoninae	Savignia sp.1	KT003165	KT002977	KT003071	KT002778	KT002879	Wang et al., 2015
Linyphiidae	Erigoninae	Savignia sp.2	KT003166	KT002978	KT003072	KT002779	KT002880	Wang et al., 2015
Linyphiidae	Erigoninae	Scotinotylus sp.A	KT003167	KT002979	KT003073	KT002780	KT002881	Wang et al., 2015
Linyphiidae	Erigoninae	Scotinotylus tianschanicus	KT003188	KT003002	KT003096	KT002803	KT002903	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa lanyuensis *	OL691622	OL691625	OL691629	OL693167	OL702838	CNU
Linyphiidae	Ipainae	Solenysa longqiensis	KT003169	KT002981	KT003075	KT002782	KT002883	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa macrodonta *	OL691623	OL691627	OL691631	OL693169	OL702840	CNU
Linyphiidae	Ipainae	Solenysa mellotteei	KT003168	KT002980	KT003074	KT002780	KT002882	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa ogatai *		OL691626	OL691630	OL693168	OL702839	CNU
Linyphiidae	Ipainae	Solenysa partibilis	KT003170	KT002983	KT003077	KT002784	KT002885	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa protrudens	KT003171	KT002984	KT003078	KT002785	KT002886	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa reflexilis	KT003172	KT002985	KT003079	KT002786	KT002887	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa retractilis	KT003174	KT002987	KT003081	KT002788	KT002889	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa tianmushana	KT003175	KT002988	KT003082	KT002788	KT002890	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa trunciformis		KT002982	KT003076	KT002783	KT002884	Wang et al., 2015
Linyphiidae	Ipainae	Solenysa wulingensis	KT003176	KT002989	KT003083	KT002790		Wang et al., 2015
Linyphiidae	Ipainae	Solenysa yangmingshana *		OL691624	OL691628	OL693166	OL702837	CNU
Linyphiidae	Stemonyphantinae	Stemonyphantes abantensis	KT003177	KT002990	KT003084	KT002791	KT002891	Wang et al., 2015
Linyphiidae	Stemonyphantinae	Stemonyphantes lineatus	FJ838691	FJ838715		FJ838667	FJ838761	Arnedo et al., 2009
Linyphiidae	Stemonyphantinae	Stemonyphantes sibiricus	FJ838692			FJ838668	FJ838762	Arnedo et al., 2009
Linyphiidae	Erigoninae	Styloctetor compar	KT003178	KT002991	KT003085	KT002792	KT002892	Wang et al., 2015
Linyphiidae	Micronetinae	Tapinopa guttata	KT003179	KT002992	KT003086		KT002893	Wang et al., 2015
Linyphiidae	Micronetinae	Tenuiphantes sp.1		KT002993	KT003087	KT002793	KT002894	Wang et al., 2015
Linyphiidae	Micronetinae	Tenuiphantes sp.2	KT003180	KT002994	KT003087	KT002794	KT002895	Wang et al., 2015
Linyphiidae	Micronetinae	Tenuiphantes tenuis	FJ838693	FJ838716		FJ838669	FJ838763	Arnedo et al., 2009
Linyphiidae	Erigoninae	Ternatus malleatus	KT003181	KT002995	KT003089	KT002795	KT002896	Wang et al., 2015
Linyphiidae	Erigoninae	Ternatus siculus	KT003182	KT002996	KT003090	KT002795	KT002897	Wang et al., 2015
Linyphiidae	Erigoninae	Ummeliata insecticeps	KT003184	KT002998	KT003092	KT002798	KT002899	Wang et al., 2015
Linyphiidae	Micronetinae	Vagiphantes vaginatus	KT003185	KT002999	KT003093	KT002799	KT002900	Wang et al., 2015
Linyphiidae	Ipainae	Wubanoides sp.	KT003186	KT003000	KT003094	KT002800	KT002901	Wang et al., 2015

3.2. Phylogeny of Solenysa

The ML trees inferred from the concatenated super-matrix and from the best partition scheme and the Bayesian tree are highly congruent as far as the major clades we concern in the present study. All analyses recovered seven major clades equivalent to those found in previous studies [4]. Given the ML estimate obtained from the partitioned analysis was significantly better than from the concatenated analysis, (log-likelihoods, −45494.13 vs. −46688.08), we reported the result from the partitioned analysis in Figure 2 (see Supplementary Material Figures S1 and S2 for node supports in ML tree and Bayesian tree). All linyphiid taxa formed a monophyletic lineage sister to the pimoid clade and were grouped into seven strongly supported (bootstrap support/Bayesian posterior = 100/1.00), named as clades A, B, C, D, E, F, and S (sensu Wang et al. [4]). The monophyly of Solenysa (clade S, 100/1.00) and its sister relationship with the species-rich clade B (100/1.00) were robustly supported (100/1.00). Relationships among the seven major lineages remain the same as those of the analyses of Wang et al. [4]. One major conflict between the phylogeny we inferred here and the phylogeny reported by [8] involved relationship between Putaoa hauaping and the Stemonyphantes species (clade E). In our results, Putaoa hauaping and the Stemonyphantes species failed to form a monophyletic clade. However, our phylogeny was strongly supported by the AU test (p = 0.99).

Figure 2. ML tree of linyphiid phylogeny with proportional branch lengths. Branches in color show the seven major clades within Linyphiidae. Taxa names in color indicate their current subfamilial placements. Thickened blue branches in clade A indicate taxa having a movable epigynum. Thickened green branches in clade B indicate taxa having a desmitracheate system. Thickened branches in clade S indicate all Solenysa taxa having both movable epigynum and an intermediate type tracheate system. Bars on branches indicate corresponding node supports of the main clades: the anterior show the maximum likelihood bootstrap (BS) and the posterior Bayesian posterior probability (PP), respectively. Bars in black indicate BS > 80%; PP > 0.95; bars in grey BS < 80%, PP < 0.95. Trees with all the node supports are included as Supplementary Material.

The thirteen Solenysa taxa were further divided into four clades (Figure 2): the longqiensis clade (clade L) including S. longqiensis and S. yangmingshana (68/0.81); the wulingensis clade (clade W) including S. wulingensis, S. retractilis, and S. tianmushana (99/1.00); the mellotteei clade (clade J) including six Japanese species, S. mellotteei, S. reflexilis, S. trunciformis, S. macrodonta, S. partibili, and S. ogatai (100/1.00); and the protrudens clade (clade P), including S. protrudens and S. lanyuensis (100/1.00). Compositions of the four clades were largely congruent with the four clades recognized in phylogenetic analysis based on morphological data, each of which was supported by several synapomorphies [12]. However, the relationships among these clades in our results were: the longqiensis clade was the most basal lineage sister to all other Solenysa clades (100/1.00); the mellotteei clade was sister to the protrudens clade (100/1.00), and the clade (mellotteei + protrudens) was sister to the wulingensis clade (95/1.00).

Mapping the characters of the epigynum with an extensible base and the desmitracheate system onto the phylogenetic framework show that the movable epigynum independently evolved multiple times in clades A and S. The Solenysa clade was distantly related with the taxon of Ipainae (Wubanoides sp.) and those micronetines also having a movable epigynum. The desmitracheate system independently evolved in clade B multiple times.

Distributions of the four Solenysa clades have different patterns (Figure 1). All species of Clade J are limited to the Japanese Archipelago; the species of clades L and P are known from the southeast coast of China and Taiwan Island; those of clade W scatter in southern China, as well as the Korean Peninsula (not sampled here). All Solenysa species have a disjunct distribution, and all the materials studied here were collected from leaf litter with high ambient humidity.

3.3. Divergence Times Estimation

Divergence times of the relevant nodes within linyphiids estimated using a relaxed molecular clock method in BEAST are provided in Table 3. The dated phylogeny is shown in Figure 3, with branches of the other six major clades collapsed and the complete chronogram is presented in Supplementary Material Figure S3.

Table 3. Divergence times of relevant nodes within linyphiids estimated using a relaxed molecular clock method in BEAST.

Node	Mean	95% HPD Lower	95% HPD Upper
1	128.80	125.14	136.35
2	116.55	104.48	128.18
3	101.67	88.88	113.61
4	90.85	72.69	108.83
5	89.22	77.24	100.88
6	79.29	67.69	90.67
7	28.62	20.09	37.98
8	22.06	13.71	31.27
9	21.99	15.74	28.85
10	18.49	12.75	24.66
11	12.34	6.29	18.98
12	3.46	1.09	6.50
13	6.35	3.10	10.10
14	6.59	4.03	9.39
15	3.46	1.80	5.27
16	2.52	1.12	4.07
17	2.56	0.99	4.33
18	0.83	0.02	1.94

TMRCA of the oldest linyphiids fossil: Exponential prior (Mean = 10, Offset = 125). TMRCA of Orsonwelles polites and Orsonwelles malus: Normal prior (Mean = 3.0 mya, SD = 0.5). Time in million years ago (mya). Nodes labeled as in Figure 3. HPD, highest posterior density; TMRCA, time of most recent common ancestor.

Figure 3. Chronogram of linyphiids. Branches and taxon names in color show the four clades within Solenysa. Branches of other six major clades collapsed. In the complete chronogram shown in microimage, two red dots indicate calibration points. Numbers in parentheses after clade names refer to the sampling numbers of the clades and species numbers represented, respectively. Numbers above branches label the divergence nodes of the seven major clades within linyphiids and internal nodes within Solenysa. Values below branches show divergence time of nodes, and node bars show confidence intervals. Three grey belts refer to K-T boundary, early and middle Oligocene, and middle Miocene and Pliocene, respectively.

The chronogram of linyphiids suggests that all the seven major lineages survived the K-T boundary. The MRCA of all linyphiids (node 1) can be traced back to the early Cretaceous (128.80 mya, 95% HPD, 125.14–136.36 mya). Clade S and clade B (node 6) diverged around 79.29 (67.69–90.67) mya in the Cretaceous. Further diversifications in the linyphiid lineages largely took place after the K-T boundary, except for the Solenysa clade. The earliest split of Solenysa species (node 7) can only be traced back to 28.62 (20.09–37.98) mya in the middle Oligocene. In the following 10 Myr until the early Miocene, all of the four Solenysa clades (nodes 8–10) have appeared. The speciation of extant species (nodes 11–17) took place largely in the middle Miocene and the Pliocene, ranging between 12.34 (6.29–18.98) mya to 2.56 (0.99–4.33) mya, before the onset of the Quaternary climatic oscillations (2.6 mya–present [53,54]). Two exceptions involved the divergence of Japanese species, between S. partibilis and S. ogatai (node 18), which occurred much recently, around 0.83 (0.02–1.94) mya and between S. macrodonta and S. reflexilis (node 16), around 2.52 (1.12–4.07) mya during the Quaternary glaciations. In contrast, diversification in clades A and B crossed the K-T boundary and continued during the whole Cenozoic.

3.4. Temporal Patterns of Linyphiids Diversification

Analysis of lineage accumulation over time using LTT plots suggested that species of clade A and clade B began to accumulate long before the K-T boundary, while the lineage number in clade S (Solenysa) did not change until the Oligocene, long after the K-T boundary (Figure 4). Both clade S and clade B experienced obvious increases in the accumulation of lineages during the Oligocene. Besides, clade S experienced a unique fast lineage-accumulating phase during the last 10 Myr.

Figure 4. Temporal patterns of linyphiid lineage diversifications. a, stepped line in color indicate species number of lineages vary through time for Solenysa clade (brown), clade A (purple), and clade B (green). The shadowed areas in the same color show 95% confidence intervals.

4. Discussion

For a long time, a temporal framework was lacking for linyphiids, the second-largest group of spiders (but see [7,8]). Through phylogenetic reconstruction and molecular dating based on sequence data of five genes for all major groups of linyphiids, we brought a time scale to this important spider group and gained some illuminating insights into the evolutionary history that gave rise to the poor species diversity of Solenysa spiders contrasted with its sister group. We further use LTT plots to demonstrate the history of Solenysa diversification.

Solenysa spiders originated in the first radiation of linyphiids and missed the second burst of speciation in this group. The chronogram shows that the MRCA of linyphiids can be traced back to the early Cretaceous (Figure 3); it might even be traced to the Jurassic [20]. All the seven major clades, including the Solenysa clade, emerged during the Cretaceous. Being generalist predators in natural ecosystems, linyphiid spiders weave horizonal sheet webs to catch prey [1]. Their web-building level varies a lot across the seven major clades (Figure 2): most taxa of clade C + D, traditionally grouped in Linyphiinae, build aerial webs at various levels of vegetation, especially often at the crown level, while the taxa of clade ME mainly formed by species of Micronetinae and Erigoninae build their webs much closer to the ground. Generally, those micronetines with dorsal spots on their abdomen usually build aerial webs at the leaf-litter surface in forests, and those of clade B, especially those erigonines having an abdomen in grey to dark black without dorsal spots, build substrate-webs close to or even on the ground. Solenysa spiders, as members of clade ME, inhabit the litter layer in forests and have a grayish abdomen without dorsal spots; they build their webs close to the ground (see the figure in [31]). These suggest that linyphiids had their first diversification, as in many other spiders, accompanied by the co-adaptative radiation of insects and angiosperms in the Cretaceous [1,11,12,55], and diversification among the seven clades were accompanied by the divergence of their web-building levels. Furthermore, the subsequent diversification within these major clades mainly flourished in the Cenozoic in general. This indicates that the species diversity of all seven clades was significantly affected by the mass extinction of the K-T boundary (65 mya, [56]); their diversifications in the Cenozoic was, in fact, the second radiation in linyphiid evolution. Unlike other major clades, diversification in the Solenysa clade has a long-time lag of more than 50 Myr in their early history. The single lineage crossed the K-T boundary, with the earliest split occurring 28.62 mya in the middle Oligocene, much later than its sister group clade B, and their sister group clade A (Figure 4), although we expanded the sampling of Solenysa taxa in the present study.

The low species diversity of Solenysa spiders might result from both the lower diversification rate than that of their sister group clade B and the long-time lag in their evolutionary history. Among all the major clades of linyphiids, only in clade B did the desmitracheate system independently evolve multiple times (Figure 2). Although its selective advantages, either the high efficiency of anteriorly extending tracheae in providing oxygen directly to the brain and legs [35,36] or the assistance of extensively branched tracheae on water retention [9,37], have never been physiologically tested in web-weaver spiders (but see the morphological test in [57]), such a tracheal system repeatedly evolved in clade B, as well as in several litter-dweller spider groups [14,58,59], which implies its selective advantage for these spiders. Furthermore, the great species diversity of clade B, especially of the distal erigonines clade, suggest that the desmitracheate system might be a key innovation that triggered the fast diversification in clade B. Accordingly, without driving by the selection advantages of the desmitracheate system, it is not a surprise that the diversification rate of Solenysa clade is not as fast as that of clade B. Nevertheless, this is not the only reason attributed to the extreme asymmetry in the species diversity between the Solenysa clade and clade B. The long time lag across the K-T boundary to the middle Oligocene makes the Solenysa clade have no origination of any further extant groups for more than half of the common historical time shared with clades A and B (Figure 4).

Being highly dependent on high-humidity environments may be a major limit impact on Solenysa spiders. Unlike book lungs, tracheal respiration in spiders does not depend on hemolymph [36,60], and intensive branched tracheae are helpful in saving water [9,37]. Nevertheless, the intermediate-type tracheal system in Solenysa has the unbranched median pair tracheae extending into the prosoma [24]. Furthermore, their living habits are usually in areas with high ambient humidity. Although building sheet-webs at litter level, Solenysa spiders failed to leave forests for more open and less humid ecosystems, such as grasslands, or even as pioneers to colonize the ecological bare grounds as those erigonines of their sister group. Accordingly, we infer that their extreme dependence on high environmental humidity is the main constraint for their distribution.

Our results show the temporal patterns of linyphiid lineage diversification were closely related to global climate changes (Figure 3 and Figure 4). Although the Paleocene Earth was commonly considered ice-free and the global climate was warm and humid [61,62], evidence has shown that the climate in Asia was dry during most of the Paleogene. There was an arid belt that existed from the western-most part to the eastern coasts, and arid and semi-arid conditions dominated in large areas of China [63]. This led to the formation of temperate grasslands and savannah ecosystems on most land at the expense of forest decline [61,62,64]. Such ecosystem transformations might have acted as a driving force that promoted the rapid diversification of micronetines (clade A) and erigonines (clade B) but might be a major constraint on the survival of Solenysa species. Nevertheless, such a dry belt retreated northwestward from the Eocene to the Oligocene [63]. Until the late Oligocene (28–24 mya), the southeast part of China, including the southeast coasts and Taiwan Island, became humid. This might have triggered the diversification of extant Solenysa spiders (28.62 mya, Figure 3). In the following Miocene (24–5.3 mya) and the Pliocene (5.3–2.6 mya), the humid belt further expanded northwards, the whole eastern part of China transformed into humid conditions. During this time, clade S experienced a unique fast lineage-accumulating phase. Therefore, such climate changes might have released the constraint from arid conditions and promoted the diversification of Solenysa in the Neogene.

Solenysa spiders display typical characters of a relict group, with low species diversity and narrow distribution [65,66,67]. The chronogram of linyphiids shows that most Solenysa species have emerged before the onset of the Quaternary glaciations (2.6 mya, [53,54]). However, all of the 15 known species have a disjunct distribution, most of them being only known from type localities that fall near the supposed Pleistocene glacial refugia in East Asia (Figure 1; [68,69,70,71,72]). This suggests that the refugia have played an important role in maintaining these Solenysa species during the glacial period. The dramatically cold climate during the glacial period would generally incur contractions of the distribution range [65,66,73,74,75]. Such an interpretation may partially explain the current disjunct distribution patterns of Solenysa species. However, it also implies that Solenysa species have failed to expand their distributional ranges as the climate became warmer in the post-glacial periods. Generally, linyphiids, as well as most other spiders, are capable of dispersal by balloon [60]. Nevertheless, long-distance dispersal by ballooning means spiders staying a long time in the air without a water supply. The survival of Solenysa spiders depends extremely on highly humid environments that not only constrain their distribution but also limit their capacity for long-time ballooning for dispersal, especially when suitable habits are segmentized. Accordingly, the current distribution pattern of Solenysa spiders might be shaped by both the locations of those refugia they survived during the Quaternary glaciations and their weak dispersal capacity.

5. Conclusions

Our results suggest that Solenysa is a Cretaceous relict group, having survived the mass extinctions at the K-T boundary and the ecosystem transition caused by the global climate changes in the Cenozoic. Its diversification was shaped by the climatic oscillations in the Cenozoic. The low species diversity of the Solenysa clade, in contrast to its sister group, is largely due to the long time lag in its early evolutionary history. Given Solenysa represents a strongly supported major clade in linyphiid multi-locus phylogeny and is supported by several synapomorphies, it warrants a subfamilial status in Linyphiidae.