Molecular Phylogenetics of the Wrens and Allies (Passeriformes: Certhioidea), with Comments on the Relationships of Ferminia

ABSTRACT The superfamily Certhioidea is distributed on four continents and while comprising relatively few species, includes forms as diverse as creepers, nuthatches, gnatcatchers, and wrens. Previous attempts to infer the phylogeny of this lineage have focused on its higher-level relationships, consequently undersampling the New World wrens. This study reports the first nearly genus-level sampling of certhioids, based on concatenated and species tree analyses of 8520 bases of DNA sequence data from six gene regions. These analyses, while failing to completely resolve basal certhioid relationships, corroborate the monophyly of a diverse New World clade of gnatcatchers, gnatwrens, and wrens, and significantly improve our understanding of wren relationships. The inferred relationships among certhioids and wrens support an Old World origin for these lineages, with dispersal of the New World clade in the mid-Miocene, suggesting expansion and early diversification of the lineage through North America. This scenario suggests a minimum of six independent dispersal events into South America in this lineage, at least some likely to have been made prior to the Pliocene.


INTRODUCTION Previous Hypotheses of Relationship within Certhioidea
The superfamily Certhioidea is a small clade of passerine birds comprising the families Sittidae (nuthatches and the wallcreeper), Certhiidae (creepers), Polioptilidae (gnatcatchers and gnatwrens), and Troglodytidae (wrens). The monophyly of this group of approximately 141 species (Gill and Donsker, 2017) was first indicated by analyses of DNA-DNA hybridization data (Sibley and Ahlquist, 1990; fig. 1). These analyses also suggested the group was most closely related to Old World warblers, bulbuls, titmice, and relatives, leading to its placement within the oscine passerine superfamily Sylvioidea. Subsequent analyses of family-level pas¬ serine nuclear sequence data questioned placement in Sylvioidea, instead suggesting recogni¬ tion at the superfamily level (Barker et al., 2002;Barker et al., 2004;Cracraft et al., 2004). More recent molecular work has consistently supported monophyly of members of this clade (Alstrom et al., 2006;Johansson et al., 2008;Fregin et al., 2012;Moyle et al., 2016;Zhao et al., 2016), although to date few studies have included representatives of all its major lineages.
As for most passerine families, this superfamily has no known morphological synapomorphies. That the close relationship of species in the group was not previously recognized is likely due to the fact that it comprises at least four major ecotypes with differing locomotory and feeding specializations that do not appear entirely concordant with phylogeny. In the hind limb, the group is split between strong graspers that can cling to branches, vertical trunks, or even sheer stone surfaces (nuthatches, the wallcreeper, and some wrens) and relatively weak graspers that cling to trunks with tail support (creepers) or are limited to branches or horizontal surfaces (gnatcatchers, gnatwrens, and some wrens). In the rostrum, the group is split among species with long narrow mandibles specialized for reaching prey in crevices (creepers, the wallcreeper, gnatwrens, and most wrens), those with short broad bills that are used to excavate prey and even nest cavities (nuthatches), and those with delicate surface gleaning bills (gnatcatchers and some wrens). This morphological and ecological diversity led early taxonomists to associate these groups with many convergently similar lineages, including the Australasian sittellas (Daphoenositta) and treecreepers (Climacteris), the Philippine creeper (Rhabdornis), the Mala¬ gasy coral-billed nuthatch (Hypositta), and the Old World warblers (Sylviidae sensu lato, now Sylviidae, Acrocephalidae, Phylloscopidae, etc.; Alstrom et al., 2006;Fregin et al., 2012).
By contrast, relatively little consensus has been reached regarding relationships within the group. Perhaps the strongest single result has been monophyly of the New World lineage of Certhioidea, comprising the Polioptilidae and Troglodytidae. Every study including both lin¬ eages (e.g., Sibley and Ahlquist 1990;Barker et al., 2004;Barker 2004;Alstrom et al., 2006;Johansson et al., 2008;Zhao et al., 2016) has found strong support for their sister-group rela¬ tionship, although Sibley and Ahlquist (1990) found the New World verdin (.Auriparus) sister to the Polioptilidae, apparently due to a lab error or sample misidentification (subsequent studies have found Auriparus related to the penduline tits; e.g., Sheldon and Gill, 1996;Johans¬ son et al., 2008). The most consistently sampled lineages other than the wrens and gnatcatcher clade (the New World Certhioidea, NWC) have been creepers (Certhia) and nuthatches (Sitta).
All three possible relationships among these two genera and the New World lineage have been recovered in various studies: NWC+Certhia (Barker et al., 2002;Moyle et al., 2016;Zhao et al., 2016), NWC+Sitta (Alstrom et al., 2006;Johansson et al., 2008), and Sitta+Certhia (Barker et al., 2004). In terms of number of data sets (the three noted above) and data set size (4155 loci in Moyle et al., 2016), the evidence appears to be in favor of a NWC+Certhia relationship.
Placement of the enigmatic genera Salpornis (the spotted creeper) and Tichodroma (the wallcreeper) relative to the NWC and Certhia remains somewhat in question: only three studies to date have included the former and only two the latter. The three studies including Salpornis have placed it either as sister to Sitta (Johansson et al., 2008) with weak support (although with strong support separating Salpornis from Certhia), or as sister to Certhia (Sibley and Ahlquist, 1990;Zhao et al., 2016) with strong support (or unevaluable support, in the case of DNA-DNA hybridization tree). The two studies to date that have placed Tichodroma phylogenetically (Sib¬ ley and Ahlquist, 1990;Zhao et al., 2016) have supported its relationship with Sitta (with strong support in the latter study), as expected by previous morphological and behavioral evaluations (reviewed in Vaurie, 1957;Sibley and Ahlquist, 1990). Excepting the placement of Auriparus, the current consensus on certhioid relationships looks essentially the same as that of Sibley and Ahlquist in 1990 (fig. 1).

Hypotheses of Relationship within Troglodytidae
The wrens are the most diverse lineage of certhioids, comprising at least 84 species (60% of the superfamily) in 19 genera (Gill and Donsker, 2017). Despite this diversity, to date only a few studies have addressed higher-level relationships within the group in any detail.
Sibley and Ahlquist sampled only eight species in as many genera ( fig. 1), and found rela¬ tively little structure among them, with Microcerculus falling out as most divergent, and a close relationship between the Carolina (Thryothorus ludovicianus) and Bewicks (Thryomanes bewickii) wrens. Overall divergence within wrens had a maximum of AT50H = 6.0, roughly suggesting a clade age of ~14 Ma (assuming 2.35 Ma/AT50H, half the value for nonpasserines; Sibley and Ahlquist, 1990). More recent work on wren relationships based on DNA sequence data has improved our understanding of wren relationships (summarized in fig. 2). Barker (2004) showed: (1) the root of the wren tree most likely lay among a grade of highly terrestrial wrens including Salpinctes, Catherpes, Hylorchilus, and Microcerculus; (2) a close relationship between the genera Cistothorus and Troglodytes; (3) a close relation¬ ship between Campylorhynchus and Thry omanes! Thryothorus ludovicianus; (4) paraphyly of Thryothorus as recognized at the time; and (5) a well-supported relationship of "Thryotho¬ rus" except the type (T. ludovicianus) with the wren genera Cyphorhinus, Henicorhina, Uropsila, and Cinnycerthia. Subsequent work by Mann et al. (2006) extending sampling of " Thryothorus" to nearly all species of the group corroborated previous results and showed that all members of the genus except the type fell into three major clades more closely related to other genera than to the type. Two of those clades had available generic names that were resurrected, and Mann et al. erected a new genus for the third. Additional work on the genus Troglodytes and allies (Rice et al., 1999;Gomez et al., 2005) has shown that: (1) the Timberline Wren (Thryorchilus browni) is a close relative of the genus, (2) the Winter Wren may best be recognized in its own genus, Nannus (although evidence for its exclusion from Troglodytes is not overwhelming); and (3) (Rice et al., 1999;Barker 2004;Martinez Gomez et al., 2005;Mann et al., 2006). related to Microbates (Barker, unpublished data). This includes samples from all four clades of the previously recognized genus "Thryothorus" (true Thryothorus, Pheugopedius, Thryophilus, and Can¬ torchilus; Mann et al., 2006), both Troglodytes sensu stricto and Nannus (the Winter Wren, which some consider generically distinct; Rice et al., 1999;Gomez et al., 2005), and the monotypic Caribbean endemic genus Ferminia (table 1), which has never before been included in a phylogenetic study.
Similar to RAG1, ZEB1 is a strongly conserved gene with a long exon (though not the sole exon, as in RAG1) that preliminary results indicate is useful in avian phylogenetics (Herreman, 2000). Generation of Molecular Data: Genomic DNA was extracted from all samples using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). DNA from the sample of Ferminia was subsequently amplified by random priming using the illustra GenomiPhi V2 amplification kit (GE Healthcare, Pittsburgh, PA) to obtain adequate amounts of high molecular weight DNA.
All loci were amplified by polymerase chain reaction using previously described primers and cycling conditions (Barker, 2004;Barker et al., 2004;Kimball et al., 2009), although some PCRs for this study were performed using GoTaq G2 Hot Start Master Mix (Promega, Madison, WI).
To obtain complete sequences for Ferminia, additional specific primers were created for all loci (appendix 1). For most taxa, ZEB1 was amplified in three fragments using the primer pairs EF31F/EF799R, EF678F/EF1356R, and dEFlx/EF1707R (or in some cases EF_3prime; fig. 3, table 2), using a touchdown cycling profile (five cycles each at 58° C, 56° C, 54° C annealing temperature, followed by 20 at 52° C annealing), and 1 minute extension times. PCR reactions were evaluated by agarose gel electrophoresis, and successful amplifications with appropriately sized products were submitted to Beckman-Coulter Genomics (Danvers, MA) for clean up and sequencing with BigDye Terminator v3.1 on an ABI 3730 automated sequencer, using external and internal primers, as necessary Individual reads were assembled and edited in Geneious v5.6.7, then exported to text for alignment and subsequent analysis.
Alignment and Phylogenetic Analyses: Sequences for each gene region were aligned using MUSCLE v3.6 (Edgar, 2004) with default parameters, then concatenated for analysis. Certhioid phylogenies were estimated using single gene, concatenation, and species tree analyses in both likelihood and Bayesian frameworks. Likelihood searches as implemented in RAxML v7.0.3 (Stamatakis, 2006) were used to estimate a concatenated gene phylogeny, with a partitioned GTR+G4 (Lanave et al., 1984;Yang, 1994) model allowing proportional branch lengths among partitions. The partitioning scheme was selected using Partitionfinder vl.1.1 (Lanfear et al., 2012), using the greedy search algorithm and BIC as an optimality criterion, and starting with 14 parti¬ tions: all codons of each protein coding gene (cytochrome b, RAG1, RAG2, and ZEB1) separately, plus both introns (FGB-I4, FGB-I7). A search for the best tree was made 10 times from random starting points, and nodal support was assessed by 1000 bootstrap replicates (Felsenstein, 1985) using the fast search option. The same partitioning scheme (and model parameterization) was used to estimate relationships for the concatenated data set using Bayesian methods as imple¬ mented in MrBayes v3.2.5, using default priors for all parameters except for branch lengths, which were set to an exponential prior with a rate of 100, in order to avoid long branch artifacts identi¬ fied in initial runs (Marshall, 2010). Bayesian methods were also used to generate gene-specific estimates of phylogeny for comparison with the combined estimate, and to assess among-gene heterogeneity in phylogenetic estimates. For each Bayesian analysis, I performed two MCMC runs each of 2-106 generations, sampling every 100. Adequate (>200) effective sample size for parameters, parameter convergence, and burn-in were determined using Tracer vl.5, and overall topological and nodal convergence was assessed using functions of the "rwty" package (Lanfear et al., 2016) in R (R Core Team, 2016). A maximum clade credibility tree was calculated from the combined output using TreeAnnotator vl.8.3.
In addition to single gene and concatenated analyses, I estimated certhioid phylogeny using Bayesian species-tree methods as implemented in *BEAST vl.8.3 (Heled and Drummond, 2010). Species tree analyses assume free recombination between loci, but no recombination within loci, essentially treating each gene rather than each site as an independent measure of species relationships (Liu et al., 2008;Heled and Drummond, 2010). Since two pairs of the six loci included in this study-FGB-I4/FGB-I7 and RAG1/RAG2-are closely physically linked (separated by 1251 and 8042 bases respectively in Taeniopygia; GenBank assembly 3.2.4, Anno¬ tation release 101), I treated these pairs as single loci in terms of topology (but not in substitu¬ tion model), yielding effectively four independent loci for the purposes of species tree analysis.
Ploidy was set as mitochondrial for cytochrome b, and autosomal for the remaining loci, based on assumed synteny with Taeniopygia. I set a Yule prior on the species tree topology, used a piecewise constant multispecies coalescent model, and assumed an uncorrelated log-normally distributed model of lineage-specific rate variation. Priors on gene-specific rates were set as exponential distributions with means of 0.1. I performed two MCMC runs each of 1-108 gen¬ erations, sampling every 5000. Run outputs were analyzed as for the concatenated and single gene analyses reported above.
It was previously shown that RAG1 and RAG2 sequences of Sitta deviate strongly from stationarity, showing an excess of GC nucleotides at third-codon positions in comparison with other passerines (Barker et al., 2002;Barker et al., 2004). I assessed base composition variation at all loci using taxon-specific goodness-of-fit tests (Gruber et al., 2007). Sequence subsets for indi¬ vidual taxa showing significant departures from the overall mean were recoded as appropriate (e.g., AGY coding for mitochondrial DNA third positions, or RY coding for nuclear gene third positions, Phillips and Penny, 2003;Gibson et al., 2005), and potentially affected analyses rerun.
Integrated Analysis with Published Data: As noted above, Zhao et al. (2016) recently published an analysis of higher-level certhioid phylogeny. This study used data largely inde- pendent of those reported here; consequently, it is possible that integrated analysis of the two data sets could yield better support for basal relationships than achieved in either. To evaluate this, I constructed the largest complete matrix possible at the generic level. This yielded a data set of six certhioid taxa (Sitta, Tichodroma, Certhia, Salpornis, Polioptila, and Nannus) and two outgroups (a chimeric "cisticolid" including sequences of Prinia and Cisticola; and Sturnus).
These taxa were sampled for a total of 10 gene regions (the six described above, plus GAPDH-111, LDH-I3, MB-I2, ODC-I6/7), for a total of 11,883 aligned base pairs. Alignments for the Zhao et al. data were performed as described above for the new data reported here. The con¬ catenated data were analyzed using partitioned maximum likelihood and Bayesian methods as described above. In addition, the data for completely sampled loci were analyzed using species tree methods as described above, recognizing six independent gene regions: CYTB, FGB (14+17), MB-I2, ODC-16/7, RAG1+RAG2, and ZEB1.

RESULTS
Data Characteristics and Single Gene Analyses: I obtained sequence for all taxa from all loci, though a handful of taxa were incomplete for some loci, including Salpornis miss-ing the 5' half of FGB-I4, three species missing 83-133 bases from FGB-I7, and Nannus miss¬ ing 323 bases from RAG1. All sequences have been submitted to GenBank (see table 1 for accessions), and the concatenated alignment is available at TreeBase (study ID 21870). The alignments obtained from these data ranged in size from 633 (FGB-I4) to 2876 (RAG1) bases in length, with 119 (FGB-I4) to 401 (CYTB) phylogenetically informative sites (table 3). In terms of percentages, ZEB1 yielded the fewest informative sites per sequenced base (4%), the other nuclear loci were approximately equivalent to one another (10%-19%), and CYTB yielded the most (38%). Bayesian phylogenetic analyses of these data sets under a uniform GTR+I+G4 parameterization revealed significant heterogeneity in substitution dynamics among these loci (table 3), as expected given their location in differing genomes (mitochondrial versus nuclear) and variation in coding status (introns versus exons). In particular, based on estimated tree lengths, mitochondrial CYTB evolved at approximately 5x (ranging from 2.8-9.1) the rate of the nuclear genes, and showed much stronger base compositional bias (table 3) (Gibson et al., 2005;Powell et al., 2013). To assess the impact of these deviations, phylogenetic analyses of data from these genes and taxa were repeated with RY (RAG1) and AGY (CYTB) coding.  fig. 4). Resolution was best for outgroup relationships and relationships among the wrens and allies. In particular, these data supported: monophyly of the Certhioidea relative to the outgroups included here, monophyly of the New World certhioids (Troglodytidae + Polioptilidae) and of the two families in the clade; and many intergeneric relationships within the Troglodytidae. By contrast, basal relationships among certhioids were not well resolved by these data, with the strongest support being for separation of Sitta from all other taxa in only 59% of bootstrap replicates of the recoded data ( fig. 4) fig. 4 for definitions), a barplot indicates the strength of support for that node found in independent analyses of each gene region using MrBayes with a GTR+I+G4 parameterization as dark bars above the abscissa, as well as the strength of support against that node (defined as the highest support for all conflicting nodes recovered; light bars below the abscissa). The estimated posterior probabilities from concatenated analysis are shown after each node number.  Salpomis as sister taxa, and placement of Salpinctes as sister to Catherpes+Hylorchilus: neither of these relationships received strong support from either the concatenated or species tree analyses.
As expected given the evidential shift from sites to loci, support for certhioid relationships in species tree analyses was generally lower than for concatenated analyses, with 25/31 bipartitions in the species tree receiving higher posterior probabilities in concatenation. However, six biparti¬ tions actually had higher support in species tree analysis, most notably including: monophyly of a clade of wrens excluding Catherpes, Hylorchilus, Microcerculus, Salpinctes, and Odontorchilus, and a sister group relationship between Henicorhina and Cyphorhinus.
Phylogenetic Hypothesis Tests: Specific a priori hypothesis testing was performed for the concatenated data (table 4). All alternative hypotheses except one were very strongly rejected (Kass and Raftery, 1995) by empirical Bayes factor comparisons with the unconstrained analysis.
The exception was monophyly of the genus Troglodytes in the broad sense (i.e., including Nan¬ nus, Thryorchilus, and Troglodytes), which had a marginal likelihood essentially indistinguishable from the unconstrained analysis.  . 7). In particular, concatenated analyses strongly supported a sister-group relationship between Salpornis and Certhia, although this relationship only had a posterior probability of 0.28 in species tree analysis, and cytochrome b strongly conflicted with this relationship (fig. 8). As found in analyses of the broader taxon sample with fewer loci (see above), monophyly of Certhioidea was strongly supported (albeit with only two outgroups), as was a sister-group relationship between the wrens (Troglodytidae) and gnatcatchers (Polioptilidae). Relationships among other certhioid lineages (Sitta and Tichodroma) remained unresolved.

Basal Certhioid Relationships
The two previous studies with adequate higher-level sampling of certhioids (Sibley and Ahlquist, 1990;Zhao et al., 2016) were completely congruent in their estimate of relation¬ ships in the group ( fig. 1). The current study does not significantly contradict previous results, and does little in the way of corroboration. In particular, concatenated analysis of the data reported here fails to recover the previously reported sister-group relationships between Tichodroma and Sitta and between Salpornis and Certhia, although the latter relationship was recovered in species tree analyses (with poor support). Perhaps the most notable contribu¬ tion to resolving certhioid relationships here is an increase in support for the sister-group relationship between Salpornis+Certhia and the New World wren/gnatcatcher clade. Although Zhao et al. (2016)  . Shown is the maximum clade credibility tree from partitioned analysis with MrBayes. Nodes with estimated posterior probabilities >0.95 from partitioned, concatenated Bayesian analysis are indicated by black circles, and those also receiving support at the same level in species tree analyses with *BEAST (coding the data as six "genes" based on close linkage of several gene regions) are indicated by gray circles with black outline. In addition, bootstrap support values (1000 fast bootstrap replicates) from partitioned concatenated analysis with RAxML are shown below each branch. Node numbers in circles correspond to barplots in figure 8.

Basal Relationships of Troglodytidae
Both the gene and taxon sampling of this study are the best to date for addressing higherlevel relationships of wrens and gnatcatchers. As expected based on previous results (reviewed above), wrens and gnatcatchers form a well-supported clade within Certhioidea. Within the wrens, there is strong support for a basal split between a small clade of wrens with terrestrial habits (Microcerculus, Salpinctes, Catherpes, and Hylorchilus; a total of eight species, termed here the geophilous wrens) and all other wrens. Previous studies (Barker, 2004;Mann et ah, 2006) were ambiguous regarding the rooting of the wren tree, with one indel in FGB-I4 (Barker, 2004: indel 6) pointing to a root at Salpinctes, a member of the geophilous wren clade. Based on analysis of nucleotide variation in the genes sampled here, it is apparent that that indel was either homoplastic or a misalignment. Reexamination of the alignment shows that the indel involves a simple 11 base pair tandem repeat that has diverged by 1 base pair in Salpinctes, driving its alignment against the second repeat unit that is identical to Salpinctes in an out¬ group, rather than alignment with other wrens.
Perhaps the only outstanding question regarding basal relationships of wrens is the place¬ ment of the South American endemic genus Odontorchilus. This involves one of only four cases of hard incongruence among genes in this data set ( fig. 5: node 18). Two genes (FGB-I4 and RAG2) strongly support placement of the genus outside the main radiation of nongeophilous wrens, whereas one gene (FGB-I7) strongly supports its placement as sister to one of the two main clades in this radiation. A third gene (RAG1) is congruent with RAG2, but with support just below 0.9 posterior probability. Odontorchilus wrens have long been recognized as distinct, in particular due to their toothed bill (for which they are named), their preferred foraging stratum in the canopy, and their simple trilled songs; consequently, no there is no clear a priori expectation for their placement anywhere within wrens. However, it is interesting to find a South American lineage placed so deeply within the family (see below).

Relationships of Nongeophilous Wrens
The nongeophilous wrens apart from Odontorchilus are divided into two well-supported clades ( fig. 4: A, B) of nearly the same number of genera (seven and eight, respectively), and species (42 and 36, respectively). Clade A comprises subtropical and tropical species best known for their singing ability and nearly ubiquitous habit of performing vocal duets (Mann et al., 2009). Clade B comprises genera with both tropical and temperate distributions, includ¬ ing three small genera (two monotypic) with only temperate species. Many more species in this clade do not perform vocal duets, and species in two genera nest in tree cavities, a behavior otherwise only known from the genus Microcercuius, which nest in tunnels in the soil.
Clade A includes three genera formerly subsumed in the genus Thryothorus, until molecular data strongly supported placement of the type of that genus (the Carolina Wren T. ludovicianus) as sister to Thryomanes, to the exclusion of all other species that had been placed in it (Barker, 2004;Mann et al., 2006). Mann et al. (2006) recognized three clades of former "Thryothorus" wrens as genera (Pheugopedius, Thryophilus, and Cantorchilus), giving a new name to the third.
This treatment of these taxa is strongly corroborated by the current study. In particular, Bayes factor comparisons strongly reject association of Thryothorus ludovicianus with the other former members of the genus, as expected given previous analyses. In addition, Bayes factors strongly reject a monophyletic origin of the three genera in which former Thryothorus species are now placed, suggesting that these species cannot be subsumed under the oldest generic name (Pheu¬ gopedius) for the sake of simplicity alone. Relationships within this clade are generally strongly supported in concatenated Bayesian but not in concatenated-likelihood or species tree analyses (figs. 4,6). Notably, the genus Pheugopedius is strongly supported (except in species tree analysis) as sister to all other genera in the group. In concatenated Bayesian analyses, Cantorchilus is strongly supported as sister to Cinnycerthia, and Thryophilus as sister to Uropsila, explaining why monophyly of former Thryothorus species is strongly contradicted by these data. Relationships of relationship to the diverse (both phenotypically and in species numbers) genus Campylorhynchus.
The remaining four genera of Clade B formed a well-supported sister clade to these three. The only strongly supported relationship among these four genera placed the genus Troglodytes (a diverse, cosmopolitan, but relatively morphologically uniform group) sister to Thryorchilus (a distinct monotypic genus of the Central American highlands). It is possible that this close rela¬ tionship is actually due to Thryorchilus falling within Troglodytes, although the one study with broader sampling of the latter suggests otherwise (Gomez et al., 2005). The remaining ambiguities lie in the relative placement of the genera Nannus, Cistothorus, and Ferminia.
The genus Nannus is the only lineage of the wren and gnatcatcher clade with species in the Old World, comprising at least three species Holarctic in distribution (Drovetski et al., 2004). Until recently, these birds were classified as a single species in the genus Trog¬ lodytes: the Winter Wren, T. troglodytes. Mitochondrial studies have consistently pointed toward a distant relationship of Nannus species to core Troglodytes, with Thryorchilus and possibly Cistothorus intervening between the two (Rice et ah, 1999;Gomez et al., 2005).
In the current study, both Cistothorus and the genus Ferminia (never before included in a molecular phylogeny) separate Nannus from Troglodytes, though neither relationship showed substantial support (figs. 4, 6). Bayes factor comparison of these results to an analysis with Troglodytes monophyly constrained strongly favored the former (table 4).
However, the marginal likelihood of an analysis with Nannus, Thryorchilus, and Troglodytes constrained as monophyletic was indistinguishable from the unconstrained analysis (table 4), indicating that the strongest signal is for monophyly of Troglodytes+Thryorchilus. Thus, these data would not contradict a classification that subsumed all three genera (Nannus, Thryorchilus, and Troglodytes) within Troglodytes, as previously done by its describer (Bangs, 1902), and some subsequent taxonomies (e.g., Paynter and Vaurie, 1960 (Garrido and Kirkconnell, 2000). The species exhibits some interesting parallels with Cistothorus, including living in a marsh habitat, construction of woven domed nests on grasses or emergent vegetation (Martinez and Martinez, 1991;Llanes Sosa and Mancina, 2002;Forneris and Martinez, 2003), and vocal similarities. Based on recordings and videos (Internet Bird Collection, http://www.hbw.com/ibc/species/zapata-wren-ferminia-cerverai; Xeno-canto, http://www.xeno-canto.org/species/Ferminia-cerverai; both accessed Sep¬ tember 2017), male Ferminia have vocal repertoires (e.g., see Xeno-canto catalog XC256894), as in many wren species (e.g., Kroodsma, 1975;Kroodsma and Verner, 1978;Molles and Vehrencamp, 1999;Logue, 2006;Bradley and Mennill, 2009), and may engage in matched coun¬ tersinging as seen in some Cistothorus (Kroodsma and Verner, 1978). At least some Ferminia songs include a series of repeated low-frequency syllables most closely matched among wrens, based on my extensive listening to wren vocalizations both in the field and in recordings, by songs of the marsh wren C. palustris (e.g., fig. 9). In terms of plumage, it is perhaps most similar to Troglodytes, with dull brownish-dun underparts and richer brown strongly barred upperparts (first figured by Brooks in Barbour, 1928, or see Forneris andMartinez, 2003, for a pho¬ tograph), a similarity noted but dismissed by Barbour in his description of the species (Barbour, 1926). Consequently, it is perhaps unsurprising to find this species in an indeterminate position relative to these two genera. Regardless of how these relationships are ultimately resolved, this mosaic pattern of phenotypic similarities and closely spaced divergences suggests relatively rapid diversification at the base of this group. NO Both spectrograms were generated using the "spectro" function of the R package "Seewave" (Sueur et al., 2008; the C. palustris recording was also filtered from 0-1.1 kHz to remove low-fre¬ quency background noise).

Biogeography and Diversification of Certhioidea
Although not well resolved phylogenetically, the earliest divergences within Certhioidea are among Old World or ancestrally Old World lineages ( fig. 10). Both Tichodroma and Salpornis are exclusively Old World, and both Certhia (Tietze et al., 2006) and Sitta (Pasquet et ah, 2014) clearly have Old World origins, despite each lineage having invaded the New World (once in Certhia and three times in Sitta). Thus, certhioids are undoubtedly Old World (pos¬ sibly north temperate) in origin, consistent with an Old World origin for the entire certhioidmuscicapoid clade and indeed for oscines as a whole Moyle et al., 2016).
While dispersal of Certhia and Sitta into the New World has not resulted in substantial diver¬ sification (a total of six species by current taxonomy, but possibly as many as 10; Manthey et al, 2011;Walstrom et al., 2012), another certhioid lineage-the ancestor of the wrens and gnatcatchers-dispersed into the New World and diversified both in species number (a total of 106 species; Gill and Donsker, 2017) (Moyle et al., 2016). The continental distribution of each lineage is shown to the right, with black fill indicating presence, and light fill absence.
Based on a secondary point calibration (Moyle et al., 2016), the New World Certhioidea (NWC) most likely dispersed from the Old World between 16.1 (stem age) and 11.8 million (crown age) years ago, the mid-Miocene ( fig. 10; this represents a minimum range, since no uncertainty was included in scaling of this tree). If this range of estimated dispersal times is accurate, it suggests a north Pacific route into the New World via Beringia, rather than a North Atlantic or Antarctic route (Sanmartin et al., 2001). No extant member of the NWC is a strong flier, and long-distance dispersal directly into South America seems unlikely. Consequently, it is likely that the NWC diversified in North America prior to independent invasion of South America by multiple lineages, as has been previously suggested for wrens (Mayr, 1946), and as seems the case for emberizoids (Barker et al., 2015). Within wrens, this is corroborated by the primarily North American distribution of the geophilous wrens (half of the first split in the Troglodytidae); however, the relatively deep placement of the South American Odontorchilus suggests early dispersal into South America (or possibly extinction of this lineage from the north; fig. 10). By contrast, all three genera of Polioptilidae are found in both North and South America: if possible, resolution of the ancestral area of this clade will require species-level as well as (most likely) extensive intraspecific sampling. Isla Socorro, although it is worth noting that species in this genus are migratory), or early closure of the isthmus, as has recently been hypothesized (Bacon et al., 2015;Montes et al., 2015;but see O'Dea et al., 2016). Complete taxon sampling of this lineage may clarify this his¬ tory, in particular the timing and directional bias of dispersal events and subsequent diversifi¬ cation rates (e.g., Barker et al., 2013;Barker et al., 2015).

ACKNOWLEDGMENTS
I thank the curators and collections managers at the American Museum of Natural History, the Biodiversity Institute (University of Kansas), the Burke Museum (University of Washing¬ ton), the Museo de Zoologia "Alfonso L. Herrera" (Universidad Nacional Autonoma de Mexico), the Field Museum, and LSU Museum of Zoology for loans of material in their care. This paper benefitted from comments by Shanta Hejmadi, Tyler Imfeld, and Michael Wells. This work was supported by a grant from the NSF Biological Sciences program, DEB-1541312.