Reappraisal of the Identity of Batrachium pekinense (Ranunculaceae) and Its Implication for Biodiversity Conservation and Management

Abstract

Because of human-induced habitat deterioration and climate change, a large number of species are threatened and even endangered. Batrachium pekinense Liang Liu, a perennial aquatic plant endemic to Beijing, was synonymized as a widely distributed species in Northern China, B. bungei Steudel, but it was later listed in the national key protected wild plants in China. Taxonomic uncertainty should be clarified especially when related species may be allocated with a limited conservation budget. In this study, we reappraised the identity of B. pekinense based on results from molecular phylogenetic analysis and 15-year field observations. Our result shows that 77% of the 65 individuals collected from various sites share identical sequences in both the nuclear ribosomal ITS and the plastid psbA-trnH markers, all samples of the two species consist of one monophyletic clade with strong support, and continuous morphological variations on the key distinguishing character, the leaf shape, are observed in the field. Integrative evidence from both our and the others’ studies supports the same identity for B. pekinense and B. bungei. Hence, we suggest excluding B. pekinense from the list of national key protected wild plants of China in its next update and advocate the integrative evidence-based taxonomy and systematics for biodiversity conservation and management.

1. Introduction

Though debates exist about the cause of the sixth mass extinction on the earth, the fact is that human-induced biodiversity loss is widely accepted [1,2,3]. Biodiversity conservation efforts at international and national scales are both performed, and many endangered species have been better protected and managed in China in recent years [4,5]. In 2021, the lists of national key protected wild animals and plants in China are both issued, which further develop biodiversity conservation. A fundamental question in biodiversity conservation is that what is the definition of species? It is hard to hold the same standard in species classification between animals and plants and even among various plant lineages. Another concern is which kind of species should be listed. The low percentage of species coverage and the species with disputable taxonomic identity included in the list of key protected organisms and the poor outcomes of threatened species with a high proportion of budget allocation may suffer criticism [6,7,8]. Considering the shortfalls in budgets for conservation, policymakers are tasked with hard decisions about how to allocate the limited resources.

Aquatic plants often exhibit a high degree of phenotypic plasticity [9], which may be induced by environmental characteristics (e.g., nutrient, light, and water) and reproductive strategies (e.g., vegetative reproduction, hybridization, and polyploidy) [10,11], and consequently, leading to controversy in species classification and endemic species’ conservation. Among them, the monophyletic group Batrachium (DC.) Gray (Ranunculaceae) is a representative example, the taxonomy of which is obscure and is considered a taxonomist’s nightmare [11,12,13]. Based on results from molecular phylogeny, it has recently been classified as a section within the genus Ranunculus L. [14]. Several species in Batrachium are well known for the simultaneous occurrence of both capillary submerged leaves and laminar floating leaves, especially those distributed in Europe [11]. Because of frequent hybridization and polyploidy, species delimitation in Batrachium is challenging. The plastid psbA-trnH and the nuclear ribosomal ITS regions (nrITS) are suggested as informative in the identification of Batrachium [12,15].

Batrachium pekinense Liang Liou, a previously reported narrowly distributed species endemic to Beijing, China, was first listed as a key protected plant in Beijing in 2008, then listed as the national key protected plant in 2021 [16]. It is reported as a new species by Liang Liu in 1980 [17], with the original description that both submerged capillary and floating laminar leaves exist, and the ultimate lobules of floating laminar leaves are 0.2–0.6 mm in width (Figure 1A). However, this kind of dimorphic leaves and the filiform leaves (Figure 1B) are distinctly different from those species with typical dimorphic leaves, which grow in Europe as mentioned above. In the taxonomic revision of Batrachium [14], the morphology of the type specimen of B. pekinense designated by Liang Liu [17] is found to be the same as that of B. hydrophilus Bunge, which was published in 1831, and they are both treated as synonyms of a widely distributed species in Northern China whose type specimen was also collected from Beijing, B. bungei Steudel [14]. Furthermore, B. bungei (Steudel) Liang Liu is also synonymized under B. bungei Steudel [14]. Recently, B. mongolicus (Krylov) Serg. was found as a new record in China, which is documented as the only species with dimorphic leaves (i.e., both broad palmate floating laminar (Figure 1C) and filiform submerged capillary leaves) in Chinese Batrachium species [18]. Hence, it is necessary to reappraise the identity of B. pekinense from the point of view of both taxonomy and biodiversity conservation and management.

In this study, a large number of samples of B. bungei and B. pekinense from Beijing were collected and sequenced, and the variation of leaf morphology was investigated based on our 15-year field observations. We aim to elucidate the taxonomic position of B. pekinense and its relationships with other congeners based on integrated evidence. Our work will also be informative for policymakers during the assessment of species endangerment and the update of the lists of national key protected organisms.

2. Materials and Methods

2.1. Field Investigation and Sampling

We embarked on extensive field investigation in the type locality of B. pekinense, the streams within the mountains near Nankou where the Badaling Great Wall is located. Furthermore, consecutive field observations including its morphological variation, distribution pattern, and habitat were performed in streams in the mountains of Beijing, especially from Haituo Mountain in the northwest to Wuling Mountain in the northeast. Samples of 46 individuals of B. pekinense and 19 individuals of B. bungei used for phylogenetic study were collected from its distribution center, the Haituo Mountain (Figure 2, detailed sample information see Appendix A) in 2014. Voucher specimens are preserved at the herbarium of Beijing Forestry University. Related sequences of B. aquatilis L. and B. trichophyllus (Chaix ex Villars) Bosch were obtained from Lumbreras et al. [15], and R. japonicus Thunb. was selected as the outgroup, whose sequences were obtained from Hörandl et al. [19] (Appendix B).

2.2. DNA Extraction, Amplification, and Sequencing

Total genomic DNA was extracted from silica gel-dried leaf tissue using a plant genomic DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) following the manufacturer’s protocol. Two regions, nrITS and psbA-trnH, were selected and amplified following Lumbreras et al. [15]. The nrITS region that comprises ITS1, 5.8S gene, and ITS2 was sequenced with the universal ITS1 and ITS4 primers. Double-stranded DNA amplifications were performed on 20 μL with 14.1 μL of ddH₂O, 2 μL of Taqbuffer, 1.6 μL of dNTP, 0.5 μL of each primer (forward and reverse), 0.3 μL of Taq-polymerase (2.5 U/μL), and 1 μL of total DNA using a Eppendorf 580BR Thermal Cycler. The amplified products were sent to the Beijing Ruibo Xingke Biotechnology Co., Ltd. in Beijing, China for sequencing. The raw sequence fragments were assembled using Sequencher v.4.1.4 (Gene-Codes Corporation, Ann Arbor, MI, USA). The sequence data were aligned using MAFFT v.7.520 software [20] with default parameters.

2.3. Phylogenetic Inference

Topological incongruence between the ribosomal and the plastid partitions was tested using the incongruence length difference (ILD) test [21]. Congruence between nrITS and psbA-trnH was examined using PAUP* for 100 replicates (heuristic search, simple addition, TBR branch-swapping), and all trees were saved in each step. Two regions were combined because the p value is 0.07 in the ILD test.

Both maximum likelihood (ML) and maximum parsimony (MP) analyses were performed for the combined data matrix. For ML analysis, the IQ-TREE 2 software (v.2.1.3) [22] was employed, with 1000 replicates for the estimation of ultrafast bootstrap values (BS_ML) [23]. For MP analysis, heuristic searches with random stepwise addition by tree bisection-reconnection (TBR) branch swapping, with the MULTrees option, were performed using the PAUP* software (v.4.0) [24]. All character states were treated as unordered and equally weighted and gaps as missing data. The bootstrap value (BS_MP) was estimated from 1000 replicates using a heuristic search with simple addition with the TBR and MULPARS options implemented [25].

3. Results

3.1. Molecular Phylogenetic Analysis

The length of nrITS and psbA-trnH were 659 and 390 bp, respectively (Datas S1 and S2). A low number of variable sites were identified, that is, eight sites in nrITS and two in psbA-trnH. After combining them and excluding the identical sequences, six individuals of B. bungei and nine of B. pekinense were kept in the IQ-TREE 2 software, and a final data matrix with a length of 982 bp was generated and used for phylogenetic analysis (Data S3). Both the ML tree and the MP tree yielded the same tree topology at the species level. Neither B. bungei nor B. pekinense was supported as monophyletic. However, all 15 samples of these two species were nested together and consisted of one strongly supported clade (BS_MP = 100%, BS_ML = 95%) (Figure 3A). The two samples of B. trichophyllus nested as monophyletic (BS_MP = 100%, BS_ML = 92%), and sisters to B. pekinense–B. bungei clade. Samples of B. aquatilis were resolved as the basal clade on the tree of the Batrachium group (Figure 3A).

3.2. Morphological Comparison of Key Characters

The breadth of terminal leaf lobes is the only difference between B. pekinense and B. bungei [17]. The type specimen of B. pekinense is collected from the stream in the hills, where the Badaling Great Wall is located. However, our field investigation shows that Haituo Mountain, which is located north of the type locality, is the distribution center of B. pekinense. The giant Haituo Mountain consists of many mountains, such as the Song Mountain and the Yudu Mountain. Furthermore, B. bungei is observed to co-exist with B. pekinense in the streams of the Haituo Mountain. After careful morphological observation and comparison, one of the key differences between the two species, the leaf morphology that includes leaf lobes and the breadth of terminal lobes, is found without a clear cut (Figure 3B). Successive morphological variation was also detected in the population at the Bai River, which is located in northeast Beijing. Leaf morphology and the breadth of blades vary randomly, which also implies the obscure morphological boundary between B. pekinense and B. bungei, and this further corroborates the results of the phylogenetic study.

4. Discussion

4.1. The Identity of Batrachium pekinense Inferred from Multiple Evidence

Because phenotypic plasticity, vegetative reproduction, hybridization, and polyploidy are common in aquatic plants, taxonomic studies are difficult, and a large number of taxonomic uncertainties need further clarification. As an example of aquatic plants, the genus Batrachium is often considered a nightmare for taxonomists [11]. Batrachium pekinense, a narrowly distributed species endemic to north Beijing, is reported as Liang Liu with definite diagnostic characteristics, both floating and submerged leaves, when compared with B. bungei, which only have submerged leaves [17,26]. On the one hand, B. mongolicus is the only species with real floating leaves in China [18] (Figure 1C). On the other hand, the relatively broad floating type of leaves designated by Liang Liu (Figure 4) are not floating but submerged in streams in the field based on our long-term field observation. Hence, just as Wiegleb et al. mentioned [11], leaf type (submerged vs floating) in B. pekinense may be misunderstood and utilized in species identification in previous literature (e.g., Flora of Beijing and Flora of China). Floating broad laminar leaves like those in Figure 1C exist in neither the type specimens (Figure 4) nor the field populations of B. pekinense, the leaves with relatively broad terminal lobes are submerged but not floating leaves.

Based on our 15-year field observation, individuals with relatively broad terminal leaf lobes type (commonly identified as B. pekinense) and capillary type (commonly identified as B. bungei) are frequently discovered in the same spot (e.g., in streams in mountains with high elevation and ponds in lowland area), and leaves with intermediate type are also found (Figure 3B). Furthermore, leaves are found with the broad terminal lobes type at the basal part of the stem and then turned gradually to the capillary type at the top on the same individual (Figure 5). Hence, the definite diagnostic character employed in B. pekinense, leaf morphology, is less applicable when distinguished from B. bungei based on our long-term and extensive field observation.

Besides morphological plasticity, hybridization and polyploidy further complicate the taxonomy of Batrachium [11,27,28]. Based on previous phylogenetic and DNA barcoding studies focusing on Batrachium [12,15], plastid psbA-trnH and nrITS markers were employed for phylogenetic inference of B. pekinense in this study. A dense sampling of B. pekinense was performed focusing on its distribution center, which covered several origins of streams in the Haituo Mountain in northwest Beijing. The result shows that all individuals of B. pekinense and B. bungei nested in one clade with strong support (Figure 3A). Genome size, chromosome number, and ploidy are other important factors in species identification in Batrachium [11]. However, the same ploidy and approximately the same genome size are reported in both B. pekinense and B. bungei [29]. It is worth noting that, though Wiegleb et al. [14] questioned the chromosome number of Chinese Batrachium species (2n = 16), which is reported by Dahlgren [30], these results are independently reported by both Xue-Hua Liu [29] and Qin-Er Yang [31] in B. pekinense and B. bungei. Furthermore, phylogenomic inferences based on genomic datasets of plastome and nuclear genome with a whole distributional range sampling scheme for B. pekinense show that samples of these two species are recovered as monophyletic with full support [29]. Hence, multiple pieces of evidence from morphological, molecular phylogenetic, genomic, and chromosomal data support the same identity for the locally endemic one, B. pekinense, and the broadly distributed one, B. bungei. Consequently, the taxonomic treatment proposed by Wiegleb et al. [14], reducing B. pekinense to a synonym of B. bungei, is supported here.

4.2. Integrative Systematics for Species Conservation and Management

Species are key components in biodiversity conservation, while the delimitation and taxonomic treatment of several species are still controversial [32]. Though challenging, accurate species identification and classification are exclusive goals of taxonomy [33]. We are approaching the natural species definition much closer than in history; more and more new species are found, while several synonyms that may make conservationists frustrated are also reported. For example, Mussaenda anomala Li (Rubiaceae), an endemic species endemic to Guangxi, China with only the type specimen, was listed as a national key protected wild plant in China in 1999. However, its unique petaloid calyx lobes are clarified as unstable characteristics [34]. Hence, it is finally reduced to a synonym of a widely distributed species, M. shikokiana Makino [35], and is consequently removed from the national list of the 2021 version. Pyrus hopeiensis Yü (Rosaceae), a critically endangered species endemic to North China that was funded a lot for its conservation [36], is reduced to a synonym of a wildly distributed species, P. ussuriensis Maxim. based on integrative evidence from both phylogenomic and morphological data [32]. In this study, the same identity is again revealed between the locally endemic species, B. pekinense and the broadly distributed species, B. bungei. It is worthy of further investigating the relationship between B. bungei and the other species with an overlapping distribution range, e.g., B. subrigidus and B. trichophyllus.

Just as the Chinese Academician De-Yuan Hong says, we need a scientific and operative species concept in biodiversity pursuits [37]. On the one hand, all wild individuals of a genus as a whole can be included in the national key protected list, especially those with obscure taxonomic identities and complicated interspecies relationships but facing strong threats, such as Cycas L., Taxus L., and Fritillaria L. [7,38]. On the other hand, we also need a scientific attitude when facing the development of taxonomy and the change of species names. We suggest excluding B. pekinense from the list of national key protected wild plants in China in the next update. During the era of multi-omics, integrative systematics proceeded quickly, and we know much better about species and their evolution [39]. Considering the limited funds and resources, critically endangered organisms with clear taxonomic identities, including a special endangered group—taxonomists [40], should be given priority for conservation by policymakers during biodiversity conservation and management. Furthermore, the lists of national or regional key protected organisms should be updated regularly, e.g., every five years. By doing this, the precious budgets and resources may be better allocated, and the species conservation and management can be adjusted timely and appropriately.

5. Conclusions

Taxonomic studies of several aquatic plants are difficult because of their morphological plasticity, hybridization, polyploidy, and so on. Batrachium pekinense, a previously thought threatened species endemic to Beijing, which is listed as a national key protected wild plant in China, is found to share the same identity with the widely distributed B. bungei, based on integrative evidence. Hence, we agree with the taxonomic treatment of Wiegleb et al. [14], which reduces B. pekinense to be a synonym of B. bungei, and suggest its deletion from the list of national key protected wild plants in China in the coming update. Considering the limited budgets and resources, conservation priority should be given to endangered but taxonomically uncontroversial species based on multiple pieces of evidence from integrative systematics.