Phylogeny and evolution of grammitid ferns

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5 Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abt. Systematische Botanik, Georg-August-. Universität, Göttingen, Untere Karspüle ...... C. Chr. & Tardieu Papua New Guinea Ranker 1766 & Trapp (COLO, UC). AY460632 AY459465.
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Ranker & al. • Phylogeny and evolution of grammitid ferns

Phylogeny and evolution of grammitid ferns (Grammitidaceae): a case of rampant morphological homoplasy Tom A. Ranker1, Alan R. Smith2, Barbara S. Parris3, Jennifer M. O. Geiger1,6, Christopher H. Haufler4, Shannon C. K. Straub1,7, & Harald Schneider5 1 University

Museum and Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, Colorado 80309, U.S.A. [email protected] (author for correspondence) 2 University Herbarium, University of California, Berkeley, California 94720, U.S.A. [email protected] 3 Fern Research Foundation, 21 James Kemp Place, Kerikeri, Bay of Islands, New Zealand. [email protected] 4 Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, Kansas 66045, U.S.A. [email protected] 5 Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abt. Systematische Botanik, Georg-AugustUniversität, Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany. [email protected] 6 Current address: Carroll College, Department of Natural Sciences, 1601 North Benton Ave., Helena, Montana 59625, U.S.A. [email protected] 7 Current address: Department of Plant Biology, 228 Plant Science Building, Cornell University, Ithaca, New York 14853, U.S.A. [email protected] We conducted phylogenetic analyses of the fern family Grammitidaceae using sequences from two cpDNA genes and from morphological characters. Data were obtained for 73 species from most recognized genera in the family. The genera Adenophorus, Ceradenia, Calymmodon, Cochlidium, Enterosora, and Melpomene were each strongly supported as being monophyletic. Other recognized genera that were not supported as monophyletic included Ctenopteris, Grammitis, Lellingeria, Micropolypodium, Prosaptia, and Terpsichore. Several previously unrecognized clades were identified, some of which are characterized by distinctive morphological features. Analyses of the distribution of morphological character states on our inferred phylogeny showed extremely high levels of homoplastic evolution for many different characters. Homoplasy for morphological characters was considerably greater than for molecular characters. Many of the characters that exhibited high levels of convergent or parallel evolution across the phylogeny are features that have been commonly used to circumscribe genera in this group (e.g., leaf blade dissection, various rhizome scale characters, and glandular paraphyses). Conversely, some of the characters that exhibited relatively low levels of homoplasy have either not been regarded as having taxonomic value or have been ignored (e.g., root insertion, rhizome scale sheen). Our data support a New World origin of Grammitidaceae, with Old World taxa generally being more evolutionarily derived. Several clades are either primarily Neotropical or primarily Paleotropical but also have a few members distributed in the opposite hemisphere. Thus, we postulate multiple, independent dispersal and colonization events in several lineages.

KEYWORDS: atpB, Grammitidaceae, homoplasy, molecular phylogeny, morphology, rbcL.

INTRODUCTION Grammitids are a primarily tropical and subtropical group of mostly epiphytic ferns (Fig. 1). The group comprises approximately 750 species and is generally characterized by green tetrahedral spores, sporangial stalks consisting of only one row of cells, and leaf traces of single vascular strands (Parris, 1990, 1998b). This group of species has been recognized at a variety of taxonomic levels, most often as a distinct family (Newman, 1840; Ching, 1940; Parris, 1990) or as a subfamily of the Polypodiaceae (Lellinger, 1989; Tryon & Tryon, 1982). Preliminary nucleotide sequence data from the rbcL gene

indicated that grammitids nest within Polypodiaceae (Hasebe & al., 1995). That finding is strongly supported by studies with more extensive taxonomic sampling using evidence from three chloroplast DNA regions (Schneider & al., 2004). In addition, the study of Schneider & al. provides strong evidence for the monophyly of the whole grammitid group and for close relationships of grammitids and Neotropical taxa of Polypodiaceae. The circumscription and monophyly of the grammitid group have seldom been questioned, but there has been little agreement on generic delimitation. For example, Parris (1990) recognized only four genera in the 415

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Fig. 1. Examples of endemic Hawaiian grammitids. A, Adenophorus hymenophylloides; B, Grammitis tenella; C, A. tamariscinus; D, A. oahuensis. Photo credits: A, A. R. Smith; B–D, T. A. Ranker.

family, whereas Copeland (1947) recognized 12, and Parris (2003) recognized 18 genera and 14 species groups. Smith (1993) recognized 10 genera of grammitids of primarily New World distribution, but only one (Lomaphlebia J. Sm.) is endemic to the New World. Murillo & Smith (2003) recently described a monotypic, Neotropical endemic genus (Luisma). Smith (1993 and references therein) used presence or absence of hydathodes (i.e., differentiated leaf epidermal cells at vein tips) as one of the main characters separating groups of Neotropical genera of grammitids. Anhydathodous genera are Ceradenia L. E. Bishop, Enterosora Baker, and Zygophlebia L. E. Bishop, whereas all other Neotropical 416

genera have obvious adaxial hydathodes (with the exception of the austral, widespread species Grammitis poeppigiana, which lacks hydathodes, and G. patagonica, which rarely has them). Paleotropical species also sometimes show this feature, but whether hydathodes are useful for circumscribing Old World species groups is still uncertain. Additional evidence, both morphological and molecular, is needed to confirm the value of this character (or any others) in distinguishing both Neotropical and Paleotropical genera. Toward this end, we conducted phylogenetic analyses of molecular and morphological data of a taxonomically and geographically broad sample of grammitids. In

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this study, we pose the following systematic, phylogenetic, and biogeographic questions: Are the variously recognized grammitid genera monophyletic? Are there unrecognized monophyletic groups of species that merit generic ranking? Do monophyletic groups segregate along Old World-New World or other biogeographical lines? Are there phylogenetically distinct lineages characterized by the presence or absence of hydathodes or other distinctive morphological features? To address these questions, we sampled sequences of two chloroplast genes, atpB and rbcL, compiled an extensive morphological dataset, and conducted phylogenetic analyses of all datasets.

MATERIALS AND METHODS Taxon sampling and DNA extraction. — Outgroup relationships of grammitids were estimated by Schneider & al. (2004). Based on that analysis we chose five polypodioids as outgroups for the present study. Samples were either collected by one of us or were generously supplied by colleagues. We attempted to sample ingroup species from as many recognized grammitid genera as possible. Appendix 1 lists general locality, collector, collection number, herbarium of deposit for voucher specimens, and GenBank accession numbers for all taxa and DNA sequences studied (see internet version of Taxon). More complete locality data and other information can be obtained from the following web site: http://www.biology.duke.edu/pryerlab/ferndb/index. html. Six species were represented by two accessions each, which allowed us to assess intraspecific variation of individual recognized species. We extracted total cellular DNA using the CTAB method of Doyle & Doyle (1987) modified by adding 3% PVP-40 and 5 mM ascorbic acid. Sample DNA concentrations were standardized to 10 mg/mL with the aid of a minifluorometer. PCR amplification and sequencing. — We PCR-amplified and sequenced two segments of the cpDNA genome: a 1311 basepair (bp) fragment of the rbcL gene, and a 1266 bp fragment of the atpB gene. Methods followed those of Ranker & al. (2003). Sequences of rbcL were obtained for all 79 samples of grammitids and the five polypodioid outgroups. Sequences of atpB were obtained for 77 samples of grammitids and the five outgroup taxa. Phylogenetic analyses of molecular data. — Sequence fragments were edited by visual inspection of electropherograms in Sequencher (Gene Codes Corp.) and consensus sequences were aligned manually in PAUP* 4.0b10 (Swofford, 1998) with no inferred gaps. We first conducted maximum parsimony (MP) phy-

Ranker & al. • Phylogeny and evolution of grammitid ferns

logenetic analyses as implemented in PAUP* 4.0b10 (Swofford, 1998) on each set of DNA sequences separately. All characters were unordered and equally weighted. Invariant characters were omitted from all analyses. For each single-gene dataset, we employed heuristic searches with 1000 random addition sequence replicates with MulTrees activated, TBR branch swapping, and ACCTRAN character-state optimization. Bootstrap analyses of single-gene datasets were conducted with 100 replicates, each with 10 random addition sequence replicates. The topologies of the strict consensus trees resulting from the two single-gene analyses were compared with Kishino-Hasegawa (KH) and ShimodairaHasegawa (SH) tests as implemented in PAUP* (Bull & al., 1993; Cunningham, 1997; Goldman & al., 2000; Schneider & al., 2004). Because the topologies did not differ statistically for either test (KH, P = 0.791; SH, P = 0.393), we conducted analyses of a combined dataset. On the combined dataset, we performed heuristic searches, as above, but with 5000 random addition sequence replicates. We also conducted a decay analysis of branch support (Bremer, 1988; Donoghue & al., 1992) on the combined dataset with AutoDecay 4.0.1 (Eriksson, 1998). We conducted a heuristic bootstrap analysis of the combined dataset in PAUP* with 100 replicates, each with 10 random addition sequence replicates. We conducted another heuristic analysis of the combined dataset, as above, but with a transition:transversion (ti:tv) weighting scheme that took into account the parsimony informativeness of transitions vs. transversions in each gene separately. We estimated ti:tv ratios by comparing the length of MP trees with transitions omitted vs. with all variable sites included (see Martin & Naylor, 1997). For rbcL we found a ti:tv ratio of 4:1, and for atpB we found a ratio of 8:1. MODELTEST version 3.06 (Posada & Crandall, 1998) was employed to find the model of DNA substitution that best fit the data. Both hierarchical likelihood ratio tests and the Akaike Information Criterion method found that the TVM+G model was the best fit, and the following settings were inserted in the PAUP* combined dataset file: Lset Base = (0.2609 0.2262 0.1815) Nst = 6 Rmat = (1.4824 8.0219 0.7070 1.0769 8.0219) Rates = gamma Shape = 3.1038 Pinvar = 0; with those settings, we conducted a heuristic maximum likelihood (ML) phylogenetic analysis in PAUP* without a molecular clock constraint and with MulTrees activated and TBR branch swapping. We also conducted a ML bootstrap analysis with the same settings and with 100 repetitions and 10 random addition sequence replicates each. Clade credibility values were estimated for the combined dataset by calculating the posterior probability for each node using Bayesian inference with a MarkovChain Monte Carlo (MCMC) sampling method as implemented in MrBayes 2.1 (Huelsenbeck & Bollback, 2001; 417

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Huelsenbeck & Ronquist, 2001) and employing the TVM+G model found by MODELTEST. A tree was sampled every 100 generations for 1,500,000 generations, and each analysis was run with four simultaneous MCMC chains. A majority-rule consensus tree was computed in PAUP* on the last 12,000 sampled trees, excluding the 3,000 trees found in the burn-in period, which was estimated following the method of Schneider & al. (2004). Posterior probability values above P = 95% were considered to be statistically significant (Rannala & Yang, 1996; Larget & Simon, 1999; Huelsenbeck & Ronquist, 2001; Lewis, 2001). Phylogenetic analyses of morphological data. — A subset of the study group comprising 69 species of grammitids (i.e., excluding four species of Adenophorus, because a more detailed analysis of the whole genus has been published elsewhere (Ranker & al., 2003) and the five polypodioid outgroup species were scored for 87 morphological and chromosomal characters (Table 1). The data matrix is available in Appendix 2 (internet version of Taxon). We conducted maximum parsimony phylogenetic analyses in PAUP* 4.0b10 (Swofford, 1998) of the morphological dataset. All characters were unordered and equally weighted. We performed a heuristic search with 100 random addition sequence replicates and with MulTrees activated, TBR branch swapping, and ACCTRAN character-state optimization. Phylogenetic analyses of combined molecular and morphological data. — We also conducted a heuristic analysis with the same conditions listed in the previous section and a bootstrap analysis each with 1000 replicates of a dataset combining the two molecular datasets and the morphological dataset. Our list of morphological characters and character states was composed from information in various monographic works, floristic accounts, and character lists for genera of Polypodiaceae (e.g., Evans, 1969; Lellinger, 1972; Roos, 1985; Hovenkamp, 1986; Hensen, 1990; Rödl-Linder, 1990, 1994a, 1994b; Bosman, 1991; Hovenkamp & Franken, 1993; Zink 1993) and Grammitidaceae (e.g., Copeland, 1952, 1956; Bishop, 1974, 1989; Parris, 1983, 1990). Studies of character analysis in Polypodiaceae (e.g., Sen & Hennipman, 1981; Hetterscheid & Hennipman, 1984; Baayen & Hennipman, 1987a, b), synoptical treatments of genera of Grammitidaceae (e.g., Bishop, 1977, 1978, 1988; Smith, 1992, 1993; Smith & Moran, 1992; Smith & al., 1991) and more comprehensive works on ferns (e.g., Tryon & Tryon, 1982; Kubitzki, 1990) were also used as sources of information about characters and character states. In addition, we examined herbarium species of each taxon in Appendix 1 in several herbaria, particularly AK and UC. Coding of the character states does not

imply a transformation series, i.e., the coding is neutral. To explore the evolution of morphological traits, we employed the strict consensus tree resulting from the heuristic MP analysis of the combined rbcL-atpB. We used MacClade 4.0 PPC (Maddison & Maddison, 2000) to optimize character-state changes onto the strict consensus tree resulting from that MP analysis of the combined molecular dataset. [NB: using topologies of either the Bayesian inference strict consensus or the ML tree did not alter our interpretations of morphological evolution; results not shown.] We employed the “all most parsimonious states at each node” resolving option.

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RESULTS Molecular data and phylogenetic analyses. — For the 79 grammitid rbcL sequences obtained, 923

bp were invariant, 388 bp were variable, and 234 bp were parsimony-informative. For the 77 ingroup sequences of atpB obtained, 928 bp were invariant, 338 bp were variable, and 243 bp were parsimony-informative. The MP analysis of the rbcL dataset found 8,021 equally parsimonious trees. A length (L) of 1152 steps, consistency index (CI) of 0.45, and retention index (RI) of 0.67 characterized each tree. The MP analysis of the atpB dataset found 53,721 equally parsimonious trees with L = 955, CI = 0.36, and RI = 0.69. The strict consensus trees from both analyses (not shown) were highly, but not completely, dichotomously resolved. Only three of the 12 genera (Adenophorus, Enterosora, and Melpomene) from which we sampled two or more species were resolved as monophyletic in both singlegene analyses; all other such genera were resolved as monophyletic by only one analysis or by neither. Bootstrap (BS) values supporting the monophyly of Adenophorus, Enterosora, and Melpomene for rbcL were 77, 94, and 100, respectively, and for atpB were 66, 58, and 100, respectively. The MP analysis of the combined rbcL-atpB dataset found 72 equally parsimonious trees (Fig. 2) with L = 2231, CI = 0.43, and RI = 0.68. Bootstrap (BS) support varied across branches of the strict consensus tree, was greater or equal to 95% at 34 of the 76 ingroup nodes, and was greater than 70% at 55 ingroup nodes. The strict consensus tree from the Bayesian analysis of the combined molecular dataset produced a nearly identical topology to that from the MP analysis (Fig. 2). At 66 of the 76 ingroup nodes, clade credibility (= posterior probability, PP) values were greater than 95%. Although the strict consensus tree from our heuristic MP analysis resolved Clades V and VI as sister taxa (see Fig. 2), there was little BS or PP support for that relationship. We conducted a SH test (as implemented in PAUP*) to compare

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Ranker & al. • Phylogeny and evolution of grammitid ferns

Table 1. Morphological characters and character states used in the phylogenetic analysis of grammitid ferns. NA = Not Applicable.

HABIT: 1 growth habit: (0) terrestrial, (1) epigaeus (in moss layers on ground), (2) epilithic, (3) epiphytic, (4) rheophytic and/or rooted aquatic. ROOTS: 2 root density: (0) = 5 roots/mm of rhizome length, as viewed from one side, (1) > 5 roots/mm; 3 hair density: (0) = 25 hairs/mm of root length, as viewed from one side, (1) > 25 hairs/mm; 4 proliferous, producing new plantlets: (0) no, (1) yes; 5 insertion [NA for taxa with radial rhizomes]: (0) dorsally and ventrally, (1) ventral side only. RHIZOMES: 6 color: (0) brown (or green in vivo), (1) glaucous or whitish; 7 symmetry: (0) radial, (1) dorsiventral; 8 shape in cross-section: (0) terete, (1) dorsiventrally flattened; 9 diameter: (0) < 2 mm, (1) 2–5 mm; 10 branching: (0) branched, (1) commonly unbranched; 11 vascular strands: (0) 1, (1) 2-9, (2) 10-20, (3) >20; 12 sclerenchyma/collenchyma sheaths around vascular bundles: (0) absent, (1) present; 13 isolated sclerenchyma strands in cortex and pith: (0) absent, (1) present; 14 sclerenchyma/collenchyma sheath in the cortex: (0) absent, (1) present. RHIZOME INDUMENT: 15 indumentum: (0) hairs only, (1) mainly scales; 16 scale color [en masse]: (0) orangish, (1) light brown/tan, (2) red-brown to brown, (3) blackish (or gray to dark gray); 17 scale iridescence: (0) no, (1) yes; 18 scale sheen: (0) dull, (1) subglossy, (2) glossy; 19 scale cell turgidity (at midscale): (0) not turgid, (1) subturgid, (2) turgid; 20 scale attachment: (0) basifixed, (1) pseudopeltately attached, (2) peltately attached; 21 scale base shape: (0) non-auriculate, (1) auriculate; 22 scale apex: (0) obtuse, (1) acute, (2) acuminate, (3) filiform; 23 scale shape: (0) round, (1) ovate [length = 2× width], (2) lanceolate, (3) linear [length 12× width]; 24 cell shape at mid-scale in scale center [often but not always correlated with character 25]: (0) isodiametric, (1) length 1–2× width, (2) length > 2× width; 25 scale orientation: (0) tightly appressed, (1) slightly spreading, (2) strongly spreading; 26 scale clathrateness: (0) uniformly colored, (2) subclathrate, (3) clathrate throughout; 27 scale margins, mid-scale: (0) entire, (1) toothed, (2) setulose, hairs eglandular, unicellular, (3) glandular, glands unbranched, (4) setulose, hairs eglandular, multicellular; 28 scale margins, apex: (0) entire, (2) setulose, hairs eglandular, unicellular, (3) glandular, glands unbranched, (4) eglandular, hairs branched; 29 scale surfaces: (0) glabrous, (1) setulose, (2) glandular. LEAVES: 30 blade termination: (0) determinate, (1) indeterminate, often with seasonal constrictions; 31 fertile-sterile leaf differentiation: (0) (nearly) monomorphic, (1) hemidimorphic - leaf tip fertile (internal dimorphism); 32 blade dissection, at least at base: (0) simple (undivided), (1) pinnatifid, (2) pinnatisect, (3) 1-pinnate, pinnae adnate, (4) pinnate-pinnatifid or more divided; 33 blade base: (0) tapered, (1) truncate; 34 blade apex: (0) gradually reduced w/confluent divisions, (1) conform to the pinnae; 35 blade tissue: (0) not spongiose, (1) spongiose; 36 sclerenchyma: (0) invisible on all parts of blade, (1) faintly visible, (2) prominently visible; 37 black blade margin: (0) absent, (1) present 38 hydathodes: (0) absent, (1) present; 39 lime dots from hydathodes: (0) absent, (1) present; 40 leaf articulation: (0) absent, (1) present; 41 pinna articulation: (0) absent, (1) present; 42 adaxial outline of stipe: (0) terete, (1) flattened, (2) sulcate with lateral ridges or flanges; 43 sclerenchyma coloration on stipe: (0) not dark-pigmented, (1) dark-pigmented; 44 stipe stele number: (0) monostele, (1) distele, (2) polystele; 45 phyllopodia: (0) absent, (1) present; 46 phyllopodia/stipe separation: (0) 0–2 rhizome width apart, (1) 2–5 rhizome width apart, (2) 5–10 rhizome width apart, (3) 10–20 rhizome width apart; 47 blade with black clavate fungal fruiting bodies (Acrospermum maxonii): (0) absent, (1) present; 48 blade with aromatic aroma (somewhat sweet and spicy) when dried: (0) absent, (1) present.

;

VENATION: 49 vein orders: (0) two, (1) three, (2) four or more; 50 secondary vein form: [NA if 2o vein are unbranched] (0) dichotomous, (1) anisotomous/non-dichotomous; 51 vein fusion in sterile blades: (0) non-anastomosing, (1) anastomosing; 52 rows of vein areoles between costa and margin: (0) 1-2, (1) 3-4, (2) 5-8; 53 vein prominence adaxially: (0) not prominent, (1) slightly prominent, (2) prominent; 54 vein prominence abaxially [raised and/or darkened]: (0) not prominent, (1) slightly prominent, (2) prominent. BLADE INDUMENT: 55 hairs: (0) absent, (1) present; 56 hair types: (0) uniseriate, unicellular, (1) uniseriate, multicellular, (2) branched; 57 branched hairs [soral paraphyses excluded]: (0) branches sessile-glandular (e;g;, Ceradenia); (1) branches hairlike, acicular, (2) branches capitate-glandular, (3) branches catenate; 58 hairs on costae and rachis adaxially: (0) absent, (1) acicular, (2) glandular, (3) branched; 59 hair color: (0) hyaline or light yellowish or pale reddish, (1) dark red brown; 60 blade scales: (0) absent, (1) present; 61 blade scale color pattern: (0) uniformly colored, (1) sharply bicolored, (2) clathrate. SORI AND INDUSIA: 62 soral receptacle: (0) (nearly) flat, (1) convex; 63 sporangial stalk width at mid-stalk: (0) 2-3 cells, (1) 1 cell; 64 sorus number per leaf: (0) >2, (1) 2 (coenosori); 65 sorus distribution: (0) throughout blade or only on proximal part, (1) confined to distal portion of blade; 66 mature sorus outline: (0) round or slightly oblong, (1) elongate, 2–10× longer than wide, (2) > 10× longer than wide; 67 sorus depth: (0) surficial, (1) sunken with sloping sides, (2) sunken with vertical sides; 68 sori embossed on adaxial surface: (0) no, (1) slightly, (2) yes, decidedly; 69 soral pit rims: (0) entirely from abaxial surface, (1) in part by blade margin; 70 soral position [applicable only when sori are uniseriate between axis and margin]: (0) nearest to penultimate axis, (1) midway between axis and margin, (2) nearer to margin than to penultimate axis; 71 sorus presence in marginal areoles (or on their bordering veins): (0) absent, (1) sometimes present, (2) always present; 72 sorus presence in costal areoles (or bordering veins): (0) absent, (1) sometimes present, (2) always present; 73 paraphyses from receptacle or disproportionately from vicinity of sorus: (0) absent, (1) present; 74 hairlike paraphyses [excluding paraphyses from sporangial walls or capsules]: (0) eglandular, (1) glandular; 75 episporangial paraphyses: (0) absent, (2) hairlike; 76 position of episporangial paraphyses: (0) from stalk, (1) from side walls of capsule, (2) from annulus; 77 localization of episporangial paraphyses at distal end: (1) scattered, (2) aggregated [as in OW Grammitis]; 78 blade margin infolded over sori: (0) no, (1) yes. SPORES: 79 color: (0) whitish, (1) yellowish, (2) brownish, (3) greenish (chlorophyllous); 80 spore number: (0) 64, (1) 32, (2) 16; 81 spore laesura: (0) linear, (1) triradiate; 82 number of cells in newly shed spores: (0) one, (1) two, (2) three to four; 83 perispore (epispore) surface: (0) (nearly) smooth or plain, (1) obviously patterned or sculptured; 84 exospore (exine) surface: (0) (nearly smooth or plain, (1) obviously patterned or sculptured. GAMETOPHYTES: 85 form: (0) cordate-thalloid, (1) unknown, (2) elongate-thalloid to filamentous; 86 rhizoid developed in the first cell division: (0) no, (1) yes. CHROMOSOMES: 87 base number: (0) 37, (1) 35, (2) 33, (3) 32.

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