Research Article |
Corresponding author: AJ Harris ( aj.harris@inbox.com ) Academic editor: Pedro Acevedo-Rodríguez
© 2017 AJ Harris, Yousheng Chen, Richard T. Olsen, Sue Lutz, Jun Wen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Harris AJ, Chen Y, Olsen RT, Lutz S, Wen J (2017) On merging Acer sections Rubra and Hyptiocarpa: Molecular and morphological evidence. PhytoKeys 86: 9-42. https://doi.org/10.3897/phytokeys.86.13532
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In this study, we expanded Acer sect. Rubra Pax to include A. sect. Hyptiocarpa section Rubra comprises two iconic species, Acer rubrum Linnaeus (red maple) and A. saccharinum Linnaeus (silver maple), of eastern North American forests as well as the rare Japanese montane species, A. pycnanthum K. Koch. Section Hyptiocarpa consists of A. laurinum Hasskarl and A. pinnatinervium Merrill, which occur in subtropical and tropical regions of southwestern China to southeast Asia. Here, we confirm prior phylogenetic results showing the close relationship between sects. Rubra and Hyptiocarpa, and we use scanning electron microscopy to demonstrate that leaves of species within these sections have similar arrangements of cuticular waxes, which account for the silvery color of their abaxial surfaces. We describe that the sections also share labile sex expression; inflorescences that range from compound racemose thyrses, to racemes or umbels and that may have undergone evolutionary reduction; and several features of their fruits, such as seed locules without keels, basal portion of wings straight, acute attachment angle between mericarps, and production of some mericarps that are seedless and partially developed at maturity. Our expansion of sect. Rubra to include sect. Hyptiocarpa better elucidates the biogeographic and evolutionary history of these species. Additionally, we show that A. laurinum and A. pinnatinervium have intergrading morphology and are probably synonymous, but we note that further studies are required to conclude their taxonomic status.
Acer laurinum , Acer pycnanthum , Acer rubrum , Acer saccharinum , cuticle, ITS, scanning electron microscopy
Acer Linnaeus, the maple genus, is remarkable for comprising 125+ species and for representing one of the largest woody plant genera in the Northern Hemisphere next to oaks and willows (
Acer and the closely-related genus, Dipteronia Oliver (2 spp.), formerly comprised Aceraceae but are now treated in tribe Acereae of Sapindaceae (
Within Acer, the circumscription of infra-generic groups has been controversial. Some groups are reasonably well agreed upon, such as section Macrantha, which includes species that have conspicuously white- or green-striped bark, and the recognition of Acer carpinifolium Sielbold & Zuccarini as the sole member of sect. Indivisia (e.g.
Another maple that has not enjoyed taxonomic stability is A. laurinum Hasskarl. Acer laurinum was described as A. javanium (Junghuhn, 1841), an impressive tree with leaves and fruits that stood out from a distance.
The affinities of sect. Hyptiocarpa remain highly speculative, and its closest relatives may be within sects. Integrifolia, Trifoliata, Rubra, or Lithocarpa (
Recently, phylogenetic studies using chloroplast and nuclear DNA with several methods of analysis have repeatedly shown strong support for the somewhat unexpected sister relationship between Acer sects. Hyptiocarpa and Rubra (
In this study, we present evidence for the relationship between sects. Rubra and Hyptiocarpa from nuclear and chloroplast phylogenies and from an analysis of leaf cuticular wax ultrastructures. We also compare other morphological features of the sections according the available literature and specimens and discuss these in the context of biogeography and evolutionary radiation. Based on the results of our study, we propose combining sects. Rubra and Hyptiocarpa sect. Rubra s.l. Throughout the study, we apply the taxonomy of
Typical specimens of Acer sects. Rubra and Hyptiocarpa, especially exhibiting leaf macromorphology. A A. rubrum B A. pycnanthum C A. saccharinum D A. laurinum. Specimens deposited at US national herbarium, and accession information visible in images. Detailed specimen records are available via the US online catalog (http://collections.nmnh.si.edu/search/botany/).
We reconstructed phylogenies of Acereae at the section-level using sequences of nuclear Internal Transcribed Spacer (ITS) and the chloroplast spacer trnD-psbM (hereafter, psbM). We selected these markers because of their utility as DNA barcodes in plants (
We obtained sequences of psbM and ITS from GenBank for representative samples of sections of Acersensu
Representative sampling of species used in this study for molecular phylogenetic analysis.
Species | GenBank- ITS | GenBank- psbM | Section affiliation sensu van Geldren ( |
Section affiliation sensu |
|
---|---|---|---|---|---|
Acereae | Acer argutum Maximowicz | AF401153 | DQ659842 | Glabra | Arguta |
Acer campestre Linnaeus | LK022558 | DQ659844 | Platanoidea | Campestria | |
Acer carpinifolium Siebold & Zuccarini | AF401148 | DQ659845 | Indivisia | Indivisa | |
Acer cissifolium (Siebold & Zuccarini) K. Koch | AY605402 | KY682748 | Negundo | Cissifolia | |
Acer distylum Siebold & Zuccarini | DQ238354 | DQ659850 | Parviflora | Distyla | |
Acer glabrum Torrey | DQ23834 | DQ659892 | Glabra | Glabra | |
Acer laurinum Hasskarl | AF241490 | DQ659854 | Hyptiocarpa | Laurina | |
Acer macrophyllum Pursh | DQ238352 | DQ659860 | Lithocarpa | Macrophylla | |
Acer negundo Linnaeus | AY605407 | DQ659864 | Negundo | Negundo | |
Acer nipponicum H. Hara | DQ366143 | DQ659865 | Parviflora | Parviflora | |
Acer palmatum Thunberg | KT160159 | DQ659867 | Palmata | Palmata | |
Acer pensylvanicum Linnaeus | AY605398 | DQ659869 | Macrantha | Macrantha | |
Acer pentaphyllum Diels | DQ238478 | DQ659870 | Pentaphylla | Acer | |
Acer pentapomicum Stewart ex Brandis | - | DQ659888 | Pubscentia | Pubscentia | |
Acer pilosum Maximowicz | DQ238345 | - | Pubscentia | Pubscentia | |
Acer platanoides Linnaeus | LK022676 | DQ659871 | Platanoidea | Platanoidea | |
Acer pseudoplatanus Linnaeus | AM238269 | DQ659872 | Acer | Acer | |
Acer pycnanthum K. Koch | AM113529 | DQ659873 | Rubra | Rubra | |
Acer rubrum Linnaeus | AJ634580 | DQ659874 | Rubra | Rubra | |
Acer saccharinum Linnaeus | AM113537 | DQ659875 | Rubra | Eriocarpa | |
Acer spicatum Lamarck | AJ634578 | DQ659879 | Parviflora | Spicata | |
Acer sterculiaceum subsp. franchettii (Pax) A.E. Murray | DQ366145 | KY682749 | Lithocarpa | Lithocarpa | |
Acer tataricum subsp. ginnala (Maximowicz) Maximowicz | AY605364 | DQ659855 | Ginnala | Trilobata | |
Dipteronia dyeriana Henry | AM182900 | DQ659838 | - | - | |
Dipteronia sinensis Oliver | AY605292 | DQ659839 | - | - | |
Outgroups | Koelreuteria Laxmann | EU72057 | DQ659835 | - | - |
Sapindus Linnaeus | AY207570 | DQ659836 | - | - | |
Xanthoceras Bunge | FJ375202 | DQ659837 | - | - |
Two sequences were new to this study: psbM of Acer sterculiaceum subsp. franchettii (Pax) A.E. Murray and A. cissifolium (Siebold & Zucc.) K. Koch. We obtained the new sequences using fresh material, which we collected from the United States National Arboretum. Our collections consisted of leaves for DNA extractions, which we preserved in silica at the time of sampling, and voucher specimens, which we deposited at the United States National Herbarium (US; http://n2t.net/ark:/65665/396759747-a431-4859-b4a7-8c57db1cc2a2 and http://n2t.net/ark:/65665/36583930c-3354-4039-9e29-f9e0f9699ecb). We performed DNA extractions using a Qiagen Plant Mini Kit (Venlo, Netherlands) according to manufacturer recommendations, and we amplified psbM using forward and reverse PCR primers from
We performed sequence alignment using the MAFFT algorithm (
Prior to phylogenetic analyses, we assessed the data matrices for base compositional heterogeneity and to determine the best nucleotide substitution model. We sought to detect base compositional heterogeneity, because it can lead to errors in phylogenetic inferences especially in the placement of outgroups and other long branches (
We performed phylogenetic analyses using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods independently for ITS and psbM as well as for the concatenated data matrix. We performed the NJ analyses in GENEIOUS TREE BUILDER using Jukes Cantor distance and 1000 NJ bootstrap (BS) replicates to assess support. We reconstructed the ML trees in MEGA 6.06 (
For the morphological study of leaves, we examined individuals representing all four species comprising Acer sects. Rubra and Hyptiocarpa. We sampled leaves from all available specimens of A. laurinum and A. pycnanthum and four specimens each of Acer rubrum and A. saccharinum (Table
Specimens of Acer sections Rubra and Hyptiocarpa from which we obtained leaf material for study. All specimens are deposited at the United States National Herbarium (US; http://collections.nmnh.si.edu/search/botany/). Locations are given as state/province, county or with as much information as is available. Refer to Table
We used a Hitachi TM300 scanning electron microscope (SEM) to examine the ultrastructure of the abaxial and adaxial surfaces of the leaves following standard protocols. We used a standard working depth of 10mm and took SEM micrographs under 15kv after determining that this intensity of the electron beam would not melt the cuticular wax. All of our scanning electron micrographs of the leaf surfaces are available from in Dryad: http://dx.doi.org/10.5061/dryad.n26nd.
Throughout, we apply the term ‘cuticle’ to all parts of the wax layer(s) above the cellulose wall of the epidermal cells. We acknowledge that the cuticle is a complex structure comprised of many well-delimited and/or intergrading components (reviewed in
We examined numerous herbarium specimens to complete this study. In particular, we examined specimens in person at US, South China Botanical Garden (IBSC), and the United States National Arboretum (NA). We also examined high resolution images of specimens online using JSTOR Global Plants (http://plants.jstor.org/) and SEINet (http://swbiodiversity.org/seinet).
The aligned sequence matrices of ITS and psbM (http://dx.doi.org/10.5061/dryad.n26nd) each had alignment scores of 0.96. The ITS matrix comprised 564 characters, and psbM had 856 characters. Neither psbM nor ITS had significant differences in base composition (χ2 crit = 10.2, p=1.0 and χ2 crit = 23.8, p=1.0, respectively).
Phylogenetic analyses of ITS showed weak support for the monophyly of the clade comprising sects. Rubra and Hyptiocarpa: NJ BS 45%, BI posterior probability (PP) 0.78, ML BS 48%. The psbM data matrix had few informative characters to distinguish a clade of sects. Rubra and Hyptiocarpa from Acer distylum Siebold & Zucc. of the monotypic sect. Distyla. Sections Rubra and Hyptiocarpa formed a trivially supported clade in the NJ phylogeny. However, a clade of sects. Rubra and Hyptiocarpa included A. distylum in the ML phylogeny. The BI results from psbM highlight the low support for the relationships among sects. Rubra, Hyptiocarpa, and Distyla in that the maximum clade credibility summary showed a clade of sects. Rubra and Hyptiocarpa, while the majority rule summary showed A. distylum included in a clade with sects. Rubra and Hyptiocarpa. The concatenated data matrix of ITS and psbM yielded moderate support for a clade of sects. Rubra and Hyptiocarpa in NJ, ML, and BI analyses. The support for the Rubra-Hyptiocarpa clade was NJ BS of 45%, BI PP of 0.74 in the maximum clade credibility tree and of 0.71 in the majority rule topology, and ML BS of 74% (Fig.
Maximum likelihood tree. Composite individuals represent sections except in the case of sects. Rubra and Hyptiocarpa, where composite individuals represent species. See Table
Our examination of leaf surface features in Acer rubrum shows that the adaxial surface bears pavement cells that are generally ovoid in shape and have wavy, jig-saw puzzle-piece-like margins (Fig.
Micrographs of the leaf surfaces of Acer rubrum. A Adaxial surface showing cell shape and organization (Thieret 22942) B Adaxial surface showing cuticle (Harris 2016-63) C, Abaxial surface showing cell shape and organization of cells and stomata (Stevens 2617) D Abaxial surface showing cuticular wax (Thieret 22942). All leaf materials are from specimens deposited at US, and parenthetical information in this legend refers to the collector name and number for the source specimen.
Wax features of leaves of Acer saccharinum are similar to those of A. rubrum. Specifically, the adaxial surface bears wavy pavement cells (Fig.
Leaves of Acer pycnanthum bears wax similar to those of A. rubrum and A. saccharinum and show wavy pavement cells with striate cuticular wax (Fig.
Micrographs of the leaf surfaces of Acer saccharinum. A Adaxial surface showing cell shape and organization (Norton 69) B Adaxial surface showing cuticle (Richardson & Robertson 915) C Abaxial surface showing cell shape and organization of cells and stomata (Brown 8023) D Abaxial surface showing cuticular wax (Coville s.n.). All leaf materials are from specimens deposited at US, and parenthetical information in this legend refers to the collector name and number.
Micrographs of the leaf surfaces of Acer pycnanthum. A Adaxial surface showing cell shape and organization (Wilson 6882) B Adaxial surface showing cuticle (Collector unknown, s.n.) C Abaxial surface showing cell shape and organization of cells and stomata (Wilson 7729) D Abaxial surface showing cuticular wax (Wilson 6882). All leaf materials are from specimens deposited at US, and parenthetical information in this legend refers to the collector name and number..
In Acer laurinum, the cuticular smooth layer on the adaxial leaf surface has wrinkles that make it appear thicker than in species of sect. Rubra s.s. The smooth layer may be slightly or extensively wrinkled across the adaxial surface (Fig.
We did not detect differences in the leaf wax features based on geographic range or, in most cases, seasonality. However, we observed one late-season Acer pycnanthum specimen with some leaves partially lacking the silvery color on the abaxial surface (Fig.
Micrographs of the leaf surfaces of Acer laurinum. A Adaxial surface showing cell shape and organization (Cult., in Hort. Bog. III,K,37) B Adaxial surface showing cuticle (Sandkuhl 21296) C Abaxial surface showing cell shape and organization of cells and stomata (Sandkuhl 21296) D Abaxial surface showing cuticular wax (Wen 13386). All leaf materials are from specimens deposited at US, parenetical information in this legend refers to the collector name and number.
Late-season specimens of Acer sect. Rubrum approaching leaf senescence. A A. pycnanthum with black arrow indicating silvery abaxial surface and green arrow indicating non- silvery surface. The inset in the upper left shows an SEM micrograph of a portion of an abaxial leaf surface from this specimen that lacks the silvery color such as the area referred to by the green arrow. Blue scale bar = 50μm. For an SEM micrograph showing a silvery portion of leaf surface from this specimen, see Figure
Our phylogenetic results are congruent with previous molecular studies, which have found well-supported close relationships between Acer sects. Rubra and Hyptiocarpa. For example,
We observed very similar cuticular wax configurations on the abaxial leaf surfaces of species of sect. Rubra and in A. laurinum. In general, these configurations comprised membranous crystals that coalesce in formations appearing as wax splatters on the surface. We unexpectedly showed evidence that cuticles comprised of membranous plates are the source of the classic silvery appearance in sects. Rubra and Hyptiocarpa by showing that when cuticular wax formation is absent in A. pycnanthum (Fig.
Some authors have speculated that cuticular wax configurations may be of limited taxonomic value, because they could vary with environment (
The cuticle layer on the adaxial surface of Acer laurinum appears less similar to the species of section Rubra. While both sects. Rubra and A. laurinum have striations, these differ in the size of the striae, or ridges, which are wider and taller in A. laurinum (compare Fig.
Acer sects. Rubra and Hyptiocarpa cannot be united strictly based on the appearance of the abaxial surfaces of their leaves. Although this feature may have taxonomic value (
Different taxonomic treatments of Hyptiocarpa do not all agree on species delimitation within the section. The large number of synonyms in Hyptiocarpa and confusion over the boundaries of species may reflect high variability and the need for additional field work to elucidate species limits or intergradation (
According to
Closer examination of Acer laurinum and A. pinnatinervium shows that they intergrade on the number of primary veins. Some collections of A. laurinum (e.g., Blume 466, L; Blume s.n., L) show strong basal acrodromous veins, while isotypes of Acer pinnatinervium (F. Kingdon-Ward 9102, A, BM) show pinnate venation with brochidodromous secondary veins near the leaf base. However, the holotype and isotype of A. laurinum (F.W. Junghuhn s.n., L, U, respectively) each show variability in venation such that some leaves have acrodromous veins and others are pinnately veined with weak brochidodromous secondaries. We also observed this variability within a specimen of A. laurinum utilized in the SEM component of this study, Cult., in Hort. Bog. III,K,37 (see Table
Leaves in sects. Rubra and Hyptiocarpa, hereafter sect. Rubrasensu latu, exhibit shapes that vary within and among species from elongate to orbicular (Fig.
Section Rubra s.l. has variable inflorescence architecture (Fig.
Species of sect. Rubra s.l. except A. pycnanthum may be monoecious or dioecious and exhibit labile sex expression among individuals (
Fruits in sect. Rubra s.l. also share many features (Fig.
Prior studies have proposed other plausible relationships for sect. Hyptiocarpa based on morphology. In particular, leaf morphology has often been used to link sect. Hyptiocarpa with Acer oblongum Wallich ex de Candolle and its close relatives in sect. Pentaphylla or Integrifolia (
Elongate leaf shape in Acer rubrum and A. pycnanthum. A–B A. rubrum C–D A. pycnanthum. Unfortunately, there is no scale for the images of A. pycnanthum, but the leaf size is similar to that illustrated in Figure
Inflorescences of Acer sects. Rubra and Hyptiocarpa. A Acer rubrum with umbels of pistilate flowers B Acer rubrum with umbels of staminate flowers C A. pycnanthum with umbels of pistilate flowers C A. saccharinum with umbels of pistilate flowers. Note flowers with two, divided persistent styles D A. pinnatinervium with racemose thyrse. Specimens in A–D deposited at US, and specimen in D deposited at the British National Museum (BM). Accession information visible in images, and detailed specimen records are available via the US online catalog (http://collections.nmnh.si.edu/search/botany/) and at the data portal of BM (http://data.nhm.ac.uk/).
Fruits of species of Acer section Rubra. A A. rubrum. Specimen on left deposited at US National herbarium (US) with collection name and number: Lilian 62. Specimen on right deposited at Kew (K) as Acer drummondii Nutt. (= A. rubrum) with collection name and number: Drummond 53. Image of fruits obtained from image of specimen deposited in JSTOR Plants (http://plants.jstor.org/) B A. pycnanthum, used with attribution to Chinese Virtual Herbarium(http://www.cvh.ac.cn/); Miyoshi Furuse 54050, PE C A. saccharinum showing fruit with two fertilized ovules (upper) compared with one fertilized ovules and one partially developed, unfertilized ovule (lower). Specimens deposited at US with collection name and number: Wolf s.n. and Pringle s.n., respectively D A. laurinum Specimen deposited at K with collection name and number: Lindley, 418. Image of fruits obtained from image of specimen deposited in JSTOR Plants. Scale bar of 1cm applies to all images.
Section Rubra s.l. may have radiated out of the tropics and into temperate areas of Japan and North America based on our phylogenetic results (Fig.
Acer sect. Hyptiocarpa W.P. Fang, Acta Phytotax. Sin. 11: 172. 1966.
Acer sect. Laurina Ogata, Bull. Tokyo Univ. Forests 63: 151. 1967.#
Acer rubrum Linnaeus.
Trees, deciduous or evergreen, with labile sex expression ranging from monoecy to dioecy (possibly exclusively dioecious in A. pycnanthum). Wood distinctly ring-porous, rays 1-4(10) cells wide. Bud scales imbricate, decussate, in pairs of 4-11. Leaves entire, unlobed, or 3- or 5- lobed, elliptic to ovate, toothed or entire, glaucous to blue-colored beneath; cuticular waxes of leaves comprising a smooth layer on the adaxial surface and bearing membranous platelets and wax splatter features abaxially; primary veins 1 or 3, 5 in 5-lobed individuals of A. saccharinum; petioles sometimes turning red (e.g., new growth, late season). Inflorescences axillary (rarely terminal) from leafless buds, usually emerging before leaves, paniculate thyrses, racemes, or umbels. Sepals 5. Petals 0 or 5, red, red-green, or green when present. Stamens 5-12, inserted on (A. laurinum and A. pinnatinervium) or outside of staminal disk, disk sometimes reduced or absent (A. rubrum, A. saccharinum, A. pycnanthum). Carpels 2. Fruits schizocarps with partially inflated seed locules, sometimes turning red during maturation, partitioning wall generally narrower than the seed locules; mericarps diverging from each other at less than 90°, wings straight to slightly convex on the proximal (vein-dense) side, curved on the distal side. Some fruits seedless and partially developed at maturity.
Five species showing a disjunct distribution between eastern and southeastern Asia (3 spp.) and eastern North America (2 spp.), a common biogeographic pattern among Northern Hemisphere plant groups (
Acer laurinum Hasskarl, Tijdschr. Natuurl. Gesch. Physiol. 10: 138. 1843.
Acer javanicum Junghuhn, 1841
Acer niveum Blume, 1847
Acer cassiifolium Blume, 1847 (as cassiaefolium)
Acer philippinum Merrill, 1906
Acer garrettii Craib, 1920
Acer decandrum Merrill, 1932
Acer chionophyllum Merrill, 1941
Acer longicarpum Hu & W. C. Cheng, 1948
Acer macropterum T. Z. Hsu & H. Sun, 1997
Acer pinnatinervium Merrill, Brittonia 4: 109. 1941.
Acer machilifolium Hu & Cheng, 1948
Acer jingdongense T. Z. Hsu, 1983
Acer pycnanthum K. Koch, Ann. Mus. Bot. Lugduno-Batavi 1: 250. 1864.
Acer rubrum Linnaeus, Sp. Pl. 1055. 1753.
Acer carolinianum Walter, 1788
Acer barbatum Michaux, 1803, pro parte
Acer sanguineum Spach, 1834
Saccharodendron barbatum (Michaux) Nieuwland, 1914, pro parte
Rufacer carolinianum (Walter) Small, 1933
Rufacer rubrum (Linneaus) Small, 1933
Acer saccharinum Linnaeus, Sp. Pl. 1055. 1753.
Acer sylvestre Young, 1783
Acer glaucum Marshall, 1785
Acer rubrum var. pallidum Aiton
Acer dasycarpum Ehrhart, 1789
Acer eriocarpum Michaux, 1803
Acer tomentosum Steudel, 1821
Acer coccineum F. Michaux
Saccharosphendamnus saccharina (Linnaeus) Nieuwland, 1914
Argentacer saccharinum (Linnaeus) Small, 1933
Based on evidence from molecular phylogeny, morphology, and leaf ultrastructure, we propose uniting sects. Rubra and Hyptiocarpa within Acer sect. Rubra. Our molecular phylogenetic results are in agreement with prior studies, which suggest that Acer sects. Rubra and Hyptiocarpa are sisters. Within these sections, species share taxonomically important characteristics including leaves with silvery abaxial surfaces resulting from similar cuticular wax structures, typically lateral inflorescences, labile sex expression, partial development of seedless fruits, and many aspects of fruit morphology. The unity of these sections yields better and more complete understanding their evolutionary and biogeographic history. We speculate that sect. Rubra s.l. radiated out of the tropics and that the radiation coincided with polyploidization.
We are indebted to Scott Whittaker of the Imaging Laboratory of the Museum of Natural History, Smithsonian Institution for assistance with all aspects of SEM.. We are also indebted to Stefan Lura and Alan Whittemore of the US National Arboretum, Harlan Svoboda of the University of Ohio, and Ricky Reyna of Athens, Ohio for assistance with collections of specimens, and Ming Kang and Hanghui Kong for providing us access to the herbarium at South China Botanical Gardens (IBSC). We received invaluable assistance from Ran Wei of Beijing Institute of Botany (PE), Rong Li of Kunming Institute of Botany (KUN), and Stefan Lura of the United States National Arboretum (NA) who provided high quality images of specimens housed at their herbaria. We also received assistance from Gerald Schoenknecht of Oklahoma State University with translation of literature from its original German and from Alan Whittemore on the taxonomic portion of this work. We are grateful to the Laboratory of Analytical Biology at the Smithsonian for assistance with DNA extractions, PCR, and sequencing. We used the Hydra supercomputer for performing phylogenetic analyses, and we are grateful to the custodians of that resource for providing us access. This project represents one outcome of a Peter Buck Postdoctoral Fellowship awarded to Harris, and the research represented collaborative work partially supported by the CAS/SAFEA International Partnership Program for Creative Research Teams.