A species of the genus Panophrys (Anura, Megophryidae) from southeastern Guizhou Province, China

Abstract Herein, we describe Panophrys congjiangensissp. nov. obtained from the Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China. Phylogenetic analyses based on the mitochondrial genes 16S rRNA and COI indicated that this new species represented an independent lineage, closely related to P. leishanensis. The uncorrected genetic distances between the new species and its closest congener, P. leishanensis, were 3.0% for 16S rRNA and 8.4% for COI. The new species is distinguished from its congeners by a combination of the following morphological characteristics (1) medium body size (SVL 28.6–33.4 mm in males and 38.4–40.2 mm in females); (2) a small horn-like tubercle at the edge of each upper eyelid; (3) the tympanum distinctly visible (TD/ED ratio 0.47–0.66); (4) vomerine teeth absent; (5) the tongue not notched behind; (6) a narrow and unobvious lateral fringe on toes; (7) relative finger lengths II < I < V < III; (8) rudimentary webs on toes; (9) hindlimbs slender, heels overlapping when thighs are positioned at right angles to the body; (10) two metacarpal tubercles on the palm, with the inner metatarsal tubercle long and oval-shaped; (11) the tibiotarsal articulation reaching the nostril when the leg is adpressed and stretched forward; (12) dorsal skin rough with numerous orange–red granules, ventral surface smooth; (13) a single internal subgular vocal sac present in males; and (14) in breeding males, weak gray-black nuptial pads with black nuptial spines present on the dorsal surface of the bases of the first and second fingers. To date, the new species is only known from the type locality.

During herpetological surveys conducted between 2019 and 2020 in Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China (Fig. 1), we captured several specimens of an unknown anuran species. Based on morphological characteristics, including body size (i.e., body length < 45 mm) and a small horn-like tubercle at the middle edge of each upper eyelid, these specimens were identified as a species of Panophrys, initially P. minor (Fei et al. 2009;Fei and Ye 2016). However, subsequent observation indicated that these newly collected specimens differed from any currently described Panophrys species. Indeed, molecular phylogenetics, comparative morphology, and bioacoustics data suggest that these specimens represent a previously unknown species. This new species is described herein.

Sampling
A total of 25 specimens were collected in this study: 22 were collected in Congjiang County, Guizhou Province, China, and were identified as an unknown species. The remaining 3 specimens, collected in Kuankuoshui National Nature Reserve, Suiyang County, Guizhou Province, China, were identified as P. jiangi. All specimens were fixed in 10% buffered formalin and later transferred to 75% ethanol for preservation. The muscle samples used for molecular analysis were preserved in 95% alcohol and stored at -20 °C. All specimens are housed at Guizhou Normal University (GZNU), Guiyang City, Guizhou Province, China.

Phylogenetic analyses
We used a total of 194 gene sequences (112 16S rRNA sequences and 82 COI sequences) for the molecular analyses, representing 102 species of subfamily Megophryinae. Two mitochondrial genes were sequenced in 10 muscle tissue samples from the specimens collected in this study, and 178 sequences were downloaded from Gen-Bank. Samples included those from the undescribed species collected and named in this study (Fig. 1). Following Mahony et al. (2017), we selected Leptobrachium boringii (Liu, 1945) and Leptobrachella oshanensis (Liu, 1950) as outgroups. The two outgroup sequences were obtained from GenBank. Details of the sequences used for phylogenetic analysis are given in Table 1. All sequences were assembled and aligned using the MUSCLE (Edgar 2004) module in MEGA 7.0 (Kumar et al. 2016) with default settings. Alignments were checked by eye and revised manually if necessary. Trimming, with gaps partially deleted, was performed using GBLOCKS 0.91b (Castresana 2000). The best-fit partitioning schemes and corresponding substitution models for the concatenated-sequence supermatrix were selected in PartitionFinder 2.1.1 using the Bayesian information criterion (Lanfear et al. 2016). As a result, the analysis suggested that the best partition scheme 16S rRNA gene/each codon position of COI gene, and selected GTR+I+G model as the best model for 16S rRNA gene, and TRNEF+G, HKY+I+G, and TIM+G model as the best model for first, second and third codons position of COI gene, respectively. Phylogenetic analysis of the concatenatedsequence matrix was performed using maximum likelihood (ML) and Bayesian inference (BI). ML and BI phylogenies based on the concatenated-sequence matrix were constructed using both IQ-tree 2.0.4 (Nguyen et al. 2015) and MrBayes 3.2.1 (Ronquist et al. 2012), according to the best-fit partitioning schemes and the corresponding substitution models. The ML analysis was performed using the best-fit model for each partition with 2000 ultrafast bootstrap (UFB) replicates (Minh et al. 2013); the analysis was continued until a correlation coefficient of at least 0.99 was reached (Hoang et al. 2018). We performed two independent BI runs using four Markov chains (three heated chains and a single cold chain). The best-fit partitioning schemes and corresponding substitution models were selected. The BI analysis started with a random tree; each run consisted of 2 × 10 7 generations, sampled every 1000 generations. Runs were considered to have converged when the average standard deviation of split frequencies (ASDSF) was less than 0.01, and the effective sample sizes (ESS) in Tracer 1.7.1 (Rambaut et al. 2018) was greater than 200. Nodes in the trees were considered wellsupported when Bayesian posterior probabilities (BPP) were ≥ 0.95 and ML ultrafast bootstrap values (UFB) were ≥ 95%. The phylogenetic trees were visualized using FigTree 1.4.3 (Rambaut 2016). The uncorrected p-distance model in MEGA 7.0 (Kumar et al. 2016) was used to calculate average genetic distances among species based on 16S rRNA and COI.

Species delimitation
To assess whether new species represent a valid species, two different methods were executed. We chose to include new species in the phylogenetic tree as well as several closely related species for species delimitation analysis. First, a Bayesian hypothesis-testing approach (Bayes Factor Delimitation, BFD) was implemented to statistically test alternate hypotheses of species delimitation (Gummer et al. 2014). Two species models were tested: 11 species (contains new species) and 10 species (lump new species with P. leishanensis). All analyses were performed in *BEAST using BEAST 1.8.2 (Drummond et al. 2012) under an uncorrelated lognormal relaxed molecular clock. A Yule process was used for the species tree prior, and the piecewise linear and constant root was used for the population size model. Two independent runs for each model were performed in BEAST 1.8.2 to assess convergence of the MCMC runs. *BEAST was run each time for 1×10 7 generations of the MCMC algorithm sampling every 1000 generations and discarding the first 25% of the iterations as "burn-in". After *BEAST analyses, two methods of marginal-likelihood estimation, including path-sampling (PS; Baele et al. 2012) and stepping-stone analysis (SS; Xie et al. 2011), were performed. PS and SS analyses were each run for a chain length of 1×10 6 generations for 100 path steps. We followed the suggestions provided by Gummer et al. (2014) to assess the strength of support for a particular species delimitation hypothesis.
In addition to the Bayesian methods tested, we also applied three tree-based species-delimitation methods, i.e., Bayesian implementation of the Poisson Tree Processes model (bPTP; Zhang et al. 2013). The parameters of these three analyses were set as follows: 1×10 5 generations, a thinning of 100 and burn-in of 10%. Convergence of models were assessed by visualizing plots of the MCMC iteration vs. the Log likelihood results. The bPTP analysis was conducted on the bPTP web server (http:// species.h-its.org/ptp/) using mtDNA-based BI gene tree as input.

Morphological comparisons
Morphometric data were collected from 19 well-preserved adult specimens (voucher information given in Table 2). Measurements were recorded to the nearest 0.1 mm with digital calipers by Tao Luo following Fei et al. (2009). A total of 27 morphological features were measured in each well-preserved specimen. These following measurements were taken:

ED
eye diameter (diameter of exposed portion of eyeball); FIL first finger length; FIIL second finger length; FIIIL third finger length; FIVL fourth finger length; FL foot length (distance from distal end of tibia to the tip of the distal phalanx of toe IV); HDL head length (from tip of snout to the articulation of the jaw); HDW head width (head width at the commissure of the jaws); HLL hindlimb length (distance from tip of fourth toe to vent); HND hand length (from the proximal border of the outer palmar tubercle to the tip of digit III); IMTL inner metatarsal tubercle length (taken as maximal length of inner metatarsal tubercle); IND internasal distance (distance between nares); IOD interorbital distance (minimum distance between upper eyelids); IPTL inner palmar tubercle length (measured as maximal distance from proximal to distal ends of the inner palmar tubercle); LAHL length of lower arm and hand (distance from the elbow to the distal end of finger IV); LW lower arm width (maximum width of the lower arm); NED nasal to eye distance (distance between the nasal and the anterior corner of the eye); OPTL outer metacarpal tubercle length (measured as maximal diameter of outer metacarpal tubercle); SNT snout length (from tip of snout to the anterior corner of the eye); SVL snout-vent length (from tip of snout to posterior margin of vent); TD tympanum diameter (horizontal diameter of tympanum); TED tympanum to eye distance (distance from anterior edge of tympanum to posterior corner of eye); TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of toe IV); THL thigh length (distance from vent to knee); TL tibia length (distance from knee to heel); TW tibia width (maximum width of the tibia); UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).
To reduce allometric effects, all measurements were size-corrected with respect to SVL prior to morphometric analysis. Principal component analyses (PCAs) of sizecorrected measurements and simple bivariate scatterplots were used to explore and characterize the morphometric differences between the new species and P. leishanensis. Mann-Whitney U tests were conducted to determine the significance of differences in morphometric characters between the new species and P. leishanensis. Mann-Whitney U tests also were used to test the significance of morphometric differences between males and females of the new species. All statistical analyses were performed using SPSS 21.0 (SPSS, Inc., Chicago, IL, USA), and differences were considered statistically significant at P < 0.05. Sex was determined based on male secondary sexual characters: the presence of a vocal sac and nuptial pads/spines (Fei and Ye 2016).
We compared the morphological characters of the new species with literature data for 59 other species in the Panophrys (Table 3). We also examined the type and/or topotype materials for P. jiangi, P. liboensis, P. shuichengensis, and P. spinata (see Appendix 1).

Bioacoustics analyses
The advertisement calls of the new species were recorded from the holotype specimen (voucher number GZNU20200706010) in the field on 5 July 2020 at the Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China. The advertisement calls were recorded in a stream, using a digital sound recorder (TASCAM DR-40) at an ambient air temperature of 25 °C and 92% humidity. Sounds were recorded within 5 cm of the calling individual. The wave-format sound files were sampled at 44 kHz, with sampling depth 24 bits. Praat 6.1.16 (Boersma 2001) was used to obtain  Table 3. References for morphological characters for congeners of the genus Panophrys.

Phylogenetic analyses and genetic divergence
ML and BI phylogenies were constructed based on two concatenated mitochondrial gene sequences: 16S rRNA (548 bp) and COI (672 bp). The ML and BI topologies were largely identical (Fig. 2). Panophrys (except for P. yeae and P. zhoui) was strongly supported as monophyletic by both phylogenetic analyses. The phylogenetic trees also supported the monophyly of four of the seven genera of subfamily Megophryinae proposed in the revision of Li et al. (2020a): Ophyrophryne, Atympanophrys, Brachytarsophrys, Panophrys (except for P. yeae and P. zhoui), and Pelobatrachus; the monophyly of Xenophrys and Ophyrophryne was not supported. In both analyses, the new species formed a lower supported clade (0.59 in BI and 56% in ML) with P. leishanensis, P. baolongensis, P. wushanensis, P. tuberogranulata, P. shimentaina, P. yangmingensis, P. jiulianensis, P. mirabilis, P. shunhuangensis, and P. acuta. However, relationships among species in this clade were not well resolved except for the following well-supported sister relationships: P. baolongensis and P. wushanensis; P. shimentaina and P. yangmingensis; and P. mirabilis and P. shunhuangensis. The new species was recovered in a relatively poorly-supported sister relationship with P. leishanensis (0.60 in BI and 79% in ML; Fig. 2). The smallest p-distance between this lineage and any other species of Panophrys was 1.2% in 16S rRNA (with P. huangshanensis) and 6.5% in COI (with P. wushanensis). These levels of divergence were similar to those between other pairs of recognized congeners. For example, the 16S rRNA p-distance was 1.2% between P. leishanensis and P. huangshanensis, 1.2% between P. jingdongensis and P. binchuanensis, while the COI p -distance was 5.9% between P. lini and P. nanlingensis, 3.6% between P. spinata and P. sangzhiensis, and 4.5% between Brachytarsophrys carinense and B. popei (Suppl. material 1: Table S1; Suppl. material 2: Table S2). These results suggested that this population, from the Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China, represented an independent evolutionary lineage.

Species delimitation
The results of the *BEAST analysis for the alternative species model are provided in Table 4. Both SS and PS estimations based on 16S rRNA+COI datasets had the largest values for the 11 species taxonomy, indicating that it was supported in favor of the currently accepted 11 species model. In addition, the results of the maximum likelihood  Table 1. solution of the bPTP analysis supported 11 species taxonomy model (Appendix 1). Thus, the results of the BFD and bPTP analyses suggest support for treating the new species as a single valid species.

Morphological analyses
The results of the Mann-Whitney U tests indicated that males of the new species differed significantly from P. leishanensis males based on several morphometric characters (all p-values < 0.05; Table 5). Using PCA, we extracted two and three principal component factors with Eigenvalues greater than two for males and females, respectively (Suppl. material 3: Table S3). The first two principal components explained 67.23% and 80.68% of the total variation in males and females, respectively. The variances in the data were mainly associated with limb and head characters, including TW, THL, HDL, LW, HDW, LAHL, HLL, FIIIL, FIL, FIIL, TFL, TL, IND, and IOD (Table 5). The characters of the new species were distinct from those of P. leishanensis on two-dimensional plots of PC1 and PC2 for both males and females (Fig. 3).
Description of holotype. GZNU20200706010 (Figs 4, 5), adult male. Medium body size, SVL 33.4 mm; head length slightly larger than head width (HDL/HDW ratio 1.02); snout short, rounded and projecting beyond the lower jaw in dorsal view, longer than eye diameter (SNT/ED ratio 1.11); nostril rounded, distinct, and closer to the tip of the snout than to the eye (SNT/NED ratio 1.83); internasal distance greater than interorbital distance (IND/IOD ratio 1.19); internasal distance greater than upper eyelid width (IND/UEW ratio 1.28); region vertical and concave; canthus rostralis well-developed; top of head slightly concave in dorsal view; a small horn-like tubercle at the edge of the upper eyelid; eyes large, slightly protuberant in dorsal view, eye diameter 34% of head length, pupils vertical (Fig. 4H); tympanum distinct, tympanum diameter less than eye diameter (TD/ED ratio 0.63); vomerine ridges and vomerine teeth absent; tongue is melon seed-shaped and not notched behind (Fig. 5E).
Forelimbs slender and comparatively short, the length of lower arm and hand 44.01% of SVL; fingers slender, relative finger lengths: II < I < IV < III; tips of fingers slightly dilated, round, without lateral fringes; one distinct subarticular tubercle at the base of each finger; two metacarpal tubercles on the palm; prominent, the outer one long and thin, the inner one oval-shaped, inner metacarpal tubercles longer than outer metacarpal tubercles (IPTL/OPT ratio 1.13). Hindlimbs slender (HLL/SVL ratio 1.80); heels slightly overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the nostril when leg stretched forward; foot length less than tibia length (FL/TL ratio 0.90); relative toe lengths I < II < V < III < IV; tips of toes round and slightly dilated; toes with narrow and unobvious lateral fringes and rudiment webs; one subarticular tubercle at the base of each toe; inner metatarsal tubercle long oval-shaped and the outer one absent.
Dorsal skin rough with numerous orange-red granules; several large warts scattered on flanks and dorsal limbs; several tubercles on upper eyelid, including a small hornlike prominent tubercle on the edge (Fig. 4H); supratympanic fold distinct; tubercles on the dorsum forming a discontinuous X-shaped ridge, the V-shaped ridges disconnected; two discontinuous dorsolateral parallel ridges on either side of the X-shaped ridges; an inverted triangular brown speckle between two upper eyelids; four transverse skin ridges on the dorsal shank and thigh; ventral surface smooth; chest with small, round glands, closer to the axilla than to midventral line; femoral glands on rear of thigh; numerous white granules on ventral surface of thigh; posterior end of body distinctly protruding, forming an arc-shaped swelling above anal region.
Coloration of holotype in life (Fig. 4). Dorsal surfaces of head and trunk brownish gray; triangular marking with light edge between eyes; dark X-shaped marking with light edge on central dorsum; supratympanic fold light brown; four dark brown transverse bands on dorsal surfaces of thigh and shank; 2-4 dark brown and white vertical bars on lower and upper lip; dark vertical band below eye; iris copper-brown; throat and anterior chest light purple-brown; belly light orange-red with large white blotch and small grey blotch in belly center, and small white blotches and large black patches on belly sides, forming a discontinuous line; ventral surfaces of forelimbs purplish brown; some white spots on the ventral surfaces of hindlimbs; palms orange-red with a small black-brown blotch; ventral surfaces of first and second toes orange-red, ventral surfaces of remaining three toes black-brown; soles black-brown; tips of digits grey-white; pectoral and femoral glands white.
Preserved holotype coloration (Fig. 5). After preservation in ethanol, dorsal surfaces light brownish grey; dorsal surface of head dark gray; X-shaped ridges on dorsum indistinct and transverse bands on limbs and digits distinct, coloration lighter; throat dark black-brown; chest light black-brown; belly light gray-white with large blackbrown blotches on sides and a small gray-brown blotch in center; posterior ventral body surface, inner thigh, and upper part of tibia milky yellow; palms and metatarsal tubercle milky yellow with a small gray-brown blotch; ventral surfaces of soles and toes dark black-brown; inner metatarsal tubercle milky yellow.
Variations. Measurements of the type series are shown in Tables 2, 4. Females (SVL 39.3 ± 0.7 mm, N = 4) had larger bodies than males (SVL 31.2 ± 1.4 mm, N = 15). In life, the diagnostic morphological characters of all paratypes were identical to those of the holotype. However, coloration and stripe patterns differed among individuals (Fig. 6). For example, male GZNU20200706007 (Fig. 6A) had a brown-black back and a black-brown belly with some large white patches, as well as two V-shaped markings that were virtually connected. This specimen also had warts on both sides of the body, forming a transverse skin ridge that almost connected to the second V-shaped marking. In contrast, male GZNU20200706008 (Fig. 6B) had a large black spot between the upper eyelids. The throat and anterior belly of this specimen were purple-brownish, while the belly was light milky yellow, with two large black blotches and a small white blotch on the body sides. In specimens GZNU20200706009 and GZNU20200706012 (Fig. 6C, E), the warts on both sides of the body formed transverse skin ridges connected to the second V-shaped marking and extending behind the tympanum; three white small blotches were present on the body sides. In specimens GZNU20200706013 and GZNU20200706012 (Fig. 6D, E), the back was light reddish brown.
Advertisement call. The call description is based on recordings of the holotype GZNU 20200706010 (Fig. 7) from the bamboo forest near the streamlet. The ambient air temperature during the recording was 25.3 °C. Each call contains 9-14 syllables (mean 11.60 ± 2.07, N = 5). The call consists of a few strophes, each 2.41-3.43 s in duration (mean 2.75 ± 0.46, N = 4). Each syllable has a duration of 0.05-0.09 s (mean 0.07 ± 0.06, N = 58). The interval between syllables has a duration of 0.10-0.31 s (mean 0.167 ± 0.042, N = 53). Sexual dimorphism. Adult males (SVL 28.6-33.4 mm) smaller than adult females . Adult males with single internal subgular vocal sac (Fig. 4A). Breeding males with grey-black nuptial pads with obvious black nuptial spines on dorsal surfaces of bases of first and second fingers.
Comparisons. Comparative data of Panophrys congjiangensis sp. nov. with 59 recognized congeners of Panophrys are given in Suppl. material 4: Table S4.

Discussion
Phylogenetic analyses based on two mitochondrial genes suggested that the specimens collected in this study fell into the Panophrys, but were distinct from all previously described species in this genus. Genetic distances between Panophrys congjiangensis sp. nov. and its sister species P. leishanensis were 3.0% for 16S rRNA and 8.4% for COI, within the ranges expected for interspecific divergences in amphibians (Fouquet et al. 2007;Che et al. 2012). Indeed, other species have been distinguished and recognized based on much lower genetic distances. For example, the p-distance is 1.2% between P. angka and P. anlongensis for 16S rRNA, and 3.6% between P. sangzhiensis and P. spinata for COI (Suppl. material 1: Table S1; Suppl. material 1: Table S2). Panophrys congjiangensis sp. nov. is morphologically similar to P. leishanensis, but Panophrys congjiangensis sp. nov. is smaller, has a narrow and unobvious lateral fringe on the toes, and the tibiotarsal articulation of the hindlimb reaches the nostril when the leg is adpressed and stretched forward. The two species can also be distinguished based on bioacoustics characters: the call of Panophrys congjiangensis sp. nov. had fewer syllables than that of P. leishanensis, and the call intervals were shorter. Without phylogenetic, morphological, and bioacoustics data, it is difficult to determine the taxonomic status of new species. In this study, these multiple pieces of evidence supported the validity of Panophrys congjiangensis sp. nov. The new species described in this study increases the number of species assigned to Panophrys to 60, with 56 recorded from China (Fei and Ye 2016;AmphibiaChina 2020;Frost 2021).
Climatic fluctuations, habitat heterogeneity, habitat diversity, and the dynamics of montane forests may play important roles in driving diversification in the Panophrys (Chen et al. 2017;Liu et al. 2018). These factors may have led to the development of complex phenotypes in this genus. Recent studies have revealed high levels of species diversity in the Panophrys (Frost 2021). However, Panophrys congjiangensis sp. nov. does not belong to any of the clades identified by Chen et al. (2017) and Liu et al. (2018), suggesting that Panophrys diversity may remain severely underestimated, even where Panophrys species are sympatric ally distributed (Li et al. 2018;Lyu et al. 2020;Su et al. 2020). Until recently, it was difficult to perform taxonomic and phylogenetic studies of the Panophrys because many species in this genus are morphologically similar and have sympatric distributions; the many possible cryptic species in the Panophrys may have hindered our understanding of diversity in this genus (Chen et al. 2017;Liu et al. 2018;Li et al. 2018;Wang et al. 2019a, b;Mahony et al. 2020;Lyu et al. 2020;Liu et al. 2020;Xu et al. 2020). The high species diversity, strong forest dependence, and sympatric distributions in the Panophrys indicate that speciation patterns, niche differentiation, and coexistence mechanisms in this genus require further study.
Biodiversity conservation in southwestern China is a priority of the Chinese government (Ministry of Environmental Protection 2015). Biodiversity conservation programs in this region play an important role in maintaining the stability of mountain ecosystems as well as protecting biodiversity (Körner and Spehn 2002;Tang et al. 2006). Mountain ecosystems are characterized by high biodiversity, with species tending to exhibit a wide range of evolutionary adaptations (McCain and Colwell 2011;Elsen and Tingley 2015). Mountain ecosystems also serve as sanctuaries for many endemic and threatened species, and thus play a major role in the maintenance of biodiversity (Favre et al. 2016). Mountains ecosystems provide key ecological service functions and provide important natural resources that are utilized by local human populations (Körner and Spehn 2002;Grêt-Regamey et al. 2012). Thus, mountain species face a higher risk of extinction due to their limited range, unique environmental adaptations, and geographic isolation, rendering mountain taxa among the most likely to be threatened by climate change.
In the past three years alone, 11 new amphibian species have been described from Guizhou Province, China Li et al. 2018a, b;Li et al. 2019a, b;Lyu et al. 2019b;Wang et al. 2019c;Wei et al. 2020;Luo et al. 2020;Liu et al. 2020;Su et al. 2020). The discovery of these new species suggests that amphibian species diversity in this region is severely underestimated. In the context of global warming, there is an urgent need for a comprehensive, systematic, and in-depth survey of the impacts of climate change on terrestrial vertebrates to provide a basis for scientific decisions regarding amphibian conservation (IPCC 2014).
Research Program B of the Chinese Academy of Sciences(CAS) (No. XDB31000000), the National Animal Collection Resource Center, China (Grant No. 2005DKA21402), the Application of Amphibian Natural Antioxidant Peptides as Cosmetic Raw Material Antioxidants (QKZYD [2020]4002). We thank Professor Paul A. Garber for editing assistance during the preparation of this manuscript. We thank LetPub (www.letpub. com) for its linguistic assistance during the preparation of this manuscript.