Unraveling the Phylogenetic Relationships of the Eccoptochilinae, an Enigmatic Array of Ordovician Cheirurid Trilobites

The Cheiruridae are a diverse group of trilobites and several subfamilies within the clade have been the focus of recent phylogenetic studies. This paper focuses on the relationships of one of those subfamilies, the Ordovician Eccoptochilinae. We analyze sixteen species from six genera within the traditionally defined group, using the pilekiid Anacheirurus frederici as an outgroup. To assess the monophyly of the Eccoptochilinae seven sphaerexochine species, Kawina arnoldi, Sphaerexochus arenosus, S. atacius, S. latifrons, S. mirus, S. parvus, and S. scabridus were included in the analysis as well. The results of this analysis show that the genus Eccoptochile represents a paraphyletic grade and species traditionally assigned to Parasphaerexochus and Skelipyx plot within Pseudosphaerexochus. Also, representative species of Sphaerexochinae plot within the traditionally defined Eccoptochilinae, suggesting Eccoptochilinae itself is paraphyletic. To resolve this, we propose all species of Pseudosphaerexochus be placed within Sphaerexochinae and Eccoptochilinae be restricted to a monotypic Eccoptochile clavigera.


Introduction
The Cheiruridae Hawle and Corda 1847 [1] are a diverse trilobite group that first appears in the Early Ordovician and persists into the Devonian. Subfamilies within this group have been the subject of recent phylogenetic studies [2][3][4] and have been useful in studying macroevolutionary patterns associated with the Ordovician mass extinction [3]. Other groups of trilobites that persisted concurrently with the cheirurids, such as the aulacopleurids, have also been useful for phylogenetic analysis and the study of paleobiogeographic patterns [5][6][7][8][9].
Lane, [10] proposed the Eccoptochilinae as a group within the Cheiruridae, and this is only one of several subfamilial classifications proposed for the Cheiruridae [11][12][13][14][15]. Lane [10] contended that the cheirurids be split into seven subfamilies, noting the wide diversity of form within the Cyrtometopinae that Ö pik [12] had used to group 14 different genera. Pä rnaste [16] agreed with Lane's assessment of the Cyrtometopinae, redefining the group based on several apomorphies and removing taxa that represented transitional forms between other groups. The Eccoptochilinae was erected by Lane based on a lack of constriction in the thoracic pleaurae (the character, which he used to remove these species from the Cyrtometopinae) as well as a prominent to effaced pitting along a transverse line across the thoracic segments (which added species from the Areiinae and a new genus, Skelipyx Lane, 1971 [10]). This new grouping included Eccoptochile Hawle and Corda, 1847 [1], Placoparina Whittard, 1940 [17], Pseudosphaerexochus Schmidt, 1881 [18], Skelipyx, and Arieaspis Pribyl and Vanek, 1964 [19].
Lane's assignment was not created within a phylogenetic framework, however, and others have speculated about the efficacy of the subfamily grouping. Pribyl et al. [20] disagreed with Lane's assessment of the group, arguing that Ö pik's [12] original grouping of the Cyrtometopinae was valid and that Lane should not have synonymized Stubblefeldia with Pseudosphaerexochus.
Whittington [21], in an attempt to address the evolutionary history of the Cheiruridae, hypothesized a theoretical phylogeny for the group. In it, Pseudosphaerexochus was grouped with members of the Sphearexochininae and Eccoptochile and Ariea are a part of a separate lineage. More recently there have been more analytical attempts to assess phylogeny within the Cheiruridae, evaluating individual subfamilies within the group. Studies of the Acanthoparyphinae, Deiphoninae, Sphaerexochinae [2][3][4] have revealed that much of the earlier understanding of the species relationships did not necessarily involve monophyletic groupings.
The purpose of this study is to resolve the phylogenetic relationships within the Eccoptochilinae, a key cheirurid subfamily needing examination in a phylogenetic framework, to test whether the clade is monophyletic and determine its position in relation to the Sphaerexochininae. Taxa analyzed include species classified by Lane [10] within the Eccoptochilinae. Further, six taxa from the Sphaerexochinae (Sphaerexochus arenosus, S. atacius, S. latifrons, S. mirus, S. parvus, S. scabridus, and Kawina arnoldi) were included to assess the monophyly and evolutionary position of the Eccoptochilinae with relation to the Spharexochinae.

Phylogenetic Analysis
Morphological terminology follows Whittington [22]. Material was examined with permission at the University of Kansas Museum of Invertebrate Paleontology (KUMIP), Naturhistoriska Riksmuseet, Stockholm, Sweden (AR) the Yale University Peabody Museum of Natural History (YPM), the Museum of Comparative Zoology, Harvard University (MCZ), the VSEGEI in Saint Petersburg, Russia, and the Paleontological Museum of the University of Oslo, Norway (PMO). All material was either loaned or studied on site.

Taxa Analyzed
Twenty-four taxa were analyzed in this phylogenetic analysis. Anacheirurus frederici Salter, 1864 [23] was used as the outgroup as it has previously been suggested [21] that the early Ordovician Pilekiidae are basal to cheirurid subfamilies such as the Eccoptochilinae. Some taxa were excluded from this analysis due to the unavailability of specimens or photographic material or because the material available was poorly preserved or lacking too many characters necessary for the analysis. These species include Eccoptochile guillieri, E. impedita, E. mariana, E. scrobiculata, E. vipera, Parasphaerexochus tuberculatus, Placoparina quadrata, Pseudosphaerexochus approximus, P. bulbosus, P. dubius, P. juvensis, P. nullicauda, P. ovalis, P. parallelus, P. pater, P. ravni, and P. wolkae. Eccoptochile tumescens was treated as E. scuticauda, following suggestions by Pribyl and Vanek [24] to synonymize the two species.

Methods
The data were analyzed using TNT v1.1 [25]. A traditional search algorithm (TBR) with 10,000 replications, 1 random seed, and 100 trees saved per replication was used to determine the most parsimonious trees for the data matrix. All characters were unweighted and all multistate characters were treated as unordered as there were no obvious criteria for ordering them. To assess tree support, bootstrap and jackknife values were calculated in TNT. Bootstrap and jackknife tests were analyzed using 10,000 replicates and a traditional search (4 characters, 10 percent of the data, were removed during the jackknife test). The matrix data were compiled into Nexus files using Mesquite v.2.75 [26], and FigTree v.1.3.1 [27] was used to generate the tree figures.

Results
Parsimony analysis recovered fourteen most parsimonious trees of length 119 steps with RI values of 0.556, and CI values (when uninformative characters are excluded) of 0.344. A strict consensus of these trees (Fig. 2.1) suggests that taxa traditionally assigned to Eccoptochile form a paraphyletic grade basal to Pseudosphaerexochus and the Sphaerexochinae. Also, Parasphaerexochus zapata and the monotypic Skelipyx cancrura fall within Pseudosphaerexochus. Areia and Placoparina plot most basally among ingroup taxa.
''Sphaerexochus'' arenosus, ''S.'' atacius, S. latifrons, S. mirus, ''S.'' parvus, S. scabridus, and Kawina arnoldi, the seven taxa chosen to represent the Sphaerexochinae do not resolve as a monophyletic clade. Based on this analysis, S. mirus, S. latifrons, and S. scabridus group together with the other four taxa creating a grade. The monophyly of this group has been discussed previously by Congreve and Lieberman [4], however these results suggest that the sphaerexochines may represent a paraphyletic grade within the traditionally defined Eccoptochilinae.

Discussion
Our analysis suggests that the traditional Eccoptochilinae is paraphyletic as the included sphaerexochine species resolved within the other ingroup taxa rather than as an independent lineage. Within the subfamily, the traditionally defined Eccoptochile forms a basal paraphyletic grade leading towards the sphaerex- Table 1. Character state distributions for taxa used in phylogenetic analysis.  4 5 6 7 8 9 10 11 1213 14 15 16 17 18 19 20 21 22 23 2425 26 27 28 29  ochines, and Parasphaerexochus zapata, Skelipyx cancrura, and various Pseudosphaerexochus species. Pseudosphaerexochus sits up the tree and is paraphyletic due to the inclusion of Parasphaerexochus and Skelipyx.
Parasphaerexochus zapata and Skelipyx cancrura are herein included within Pseudosphaerexochus to make that genus monophyletic. In addition, Pseudosphaerexochus is removed from Eccoptochilinae and reassigned to Sphaerexochinae based on the phylogenetic position of the sphaerexochine taxa included in this analysis.
Regarding Lane's original character diagnosis for the group, lack of constriction in the thoracic pleurae appears to hold true for the Eccoptochilinae and the taxa grading towards it, and we also see some evidence for this among Pseudosphaerexochus as demon- strated by P. octolobatus, however much of the other taxa within the genus are missing thoracic data to make an assessment of this character's behavior. Also, it is interesting to note that the pitting along the thorax is present in all ''Eccoptochilinae'' and absent in all sphaerexochines (with the exception of P. cancrura).
The potential paraphyly within parts of Sphaerexochus is to be noted. Congreve and Lieberman [4] had shown that the genus was monophyletic when included in an analysis with species of Kawina. By including the Eccoptochilinae with representatives from this group, our analysis suggests that Pseudosphaerexochus is a derived  [26] with genera labeled and paraphyletic genus identified using quotations following Wiley [27]. The following nodes of the tree were supported by the following jackknife confidence values (see text for jackknife procedure utilized): Node 2 = 100; Node 3 = 78; Node 4 = 92; Node 5 = 49; Node 6 = 30; Node 7 = 30; Node 8 = 42; Node 9 = 56. The following nodes of the tree were supported by the following bootstrap confidence values (see text for bootstrapping procedure utilized): Node 2 = 100; Node 3 = 29; Node 4 = 57; Node 6 = 7; Node 7 = 2; Node 8 = 8; Node 9 = 18.; 2, One of fourteen most parsimonious trees of length 119 steps. sphaerexochine and thus parts of Sphaerexochus may not be a monophyletic clade as previously thought. We will not attempt to further revise the taxonomy for this genus as it is not the main focus of this paper and will require further detailed phylogenetic analysis, however it is interesting to note that the subgenus S. (Sphaerexochus) does resolve monophyletically, consistent with the results of Congreve and Lieberman [4].
Ö pik's [12] treatment of these groups belonging to a larger Cyrtometopinae appears to be invalid. Our placement of the Sphaerexochinae within Lane's Eccoptochilinae demonstrates this and provides support for his claims that the Cyrtometopinae had a great range in morphological variation.
Further, our study provides results contradictory to what Whittington [21] had hypothesized for cheirurid relationships. Whittington saw Eccoptochile and Areia as constituents of a lineage separate from Kawina, Sphaerexochus, and Pseudosphaerexochus. The analysis supports his hypothesis that Areia is basal to Eccoptochile, however our results suggest Sphaerexochinae derived from these trilobites. Our analysis also disagrees with his suggestion that Kawina and Sphaerexochus form a separate lineage from Pseudosphaerexochus, as our tree indicates that Pseudosphaerexochus is a more derived genus that evolves out of Sphaerexochus.

Diagnosis
Genae are flat. S2 and S3 are strongly incised and as distinct as S1. S1 furrow is S-shaped and does not penetrate SO. 12 thoracic segments with transverse rows of pitting. The pygidium is shieldlike with a small terminal axial piece present.

Discussion
Because the phylogenetic analysis indicates the traditional Eccoptochile is paraphyletic, we redefine it as a monotypic taxon consisting of the type species E. clavigera. All other species originally placed within the genus Eccoptochile are placed within ''Eccoptochile'' sensu Wiley [28].

Diagnosis
Glabella is wide, hides anterior cephalic boarder in dorsal view, with curved lateral margins. Genae are strongly tilted ventrally. The anterior most position of the eye is abaxial of S2. Pitting on the thoracic segment is absent and the first axial ring of the pygidium is wide. The terminal axial piece is absent.

Discussion
To create a monophyletic genus, Parasphaerexochus zapata and Skelipyx cancrura are subsumed within Pseudosphaerexochus. These taxa share many characters with other members of Pseudosphaerexochus that support their placement within the genus. These include a Ushaped S1, a flat pygidium, pleural spines that separate from each other distally, and an absent terminal axial piece. Further, Pseudosphaerexochus is removed from Eccoptochilinae and placed within Sphaerexochinae.
Lane's diagnosis for the genus includes an inflated and ovate glabella with small cheeks and three pairs of lateral furrows, the posterior pair being most distinct. These characters are still valid for describing Pseudosphaerexochus, however they are also common among Sphaerexochus taxa as well. Lane also noted the short rounded terminal axial piece present in Pseudosphaerexochus. This analysis shows that this character was lost within the group with the exception of P. ekphyma, which plots more basally to the rest of the group and closer to Sphaerexochus. Further, Pribyl et al.'s [20] suggestion that there are two lineages within Pseudosphaerexochus based on two pygidial morphotypes does not hold true for our results.
In creating the genus Skelipyx, Lane distinguished it from Pseudosphaerexochus based on its rounder glabella, much of which is vertical or overhangs. We found the steepness of the lateral margins of the glabella to be very similar between the two genera and that degrees of roundness do not appear to be diagnosably distinct. Lane further notes the unique shape of the pygidium with the wide space between the posterior pair of spines. This character is indeed unique to this taxon, however due to its autapomorphic nature it is not included in this phylogenetic analysis. The placement of Skelipyx within Pseudosphaerexochus is consistent with Pribyl et al. [20] who assumed Skelipyx was derived from that genus.

Evolutionary Implications
It is interesting to note that, save for the one clade of trilobites belonging to the genus Sphaerexochus, all of the other species are restricted to the Ordovician. Furthermore, the early Ordovician species of Kawina and Sphaerexochus represent the only Laurentian forms, with nearly all other species of ''Eccoptochilinae'' originating in Avalonia, Bohemia, and Baltica. The topology of our analysis suggests that there may have been a dispersal event early on during the Ordovician that gave rise to the split between Sphaerexochus and Pseudosphaerexochus. In turn, these Laurentian forms would go on to diversify and dramatically expand their ranges during the Late Ordovician mass extinction [4], while all of the other ''Eccoptochilinae'' went entirely extinct. It is possible that dispersal to Laurentia may have been an important factor contributing to the group's survival. A similar pattern of survivability can be found in the homalonotid trilobites during that time period; most old world homalonotid trilobites went extinction but the one clade that dispersed to Laurentia thrived [39].