APPENDIX 3 TRICHOPTERA SPECIES HIGHLIGHTED BY BARCODE ANALYSIS IN

3 APPENDIX 1 DEVELOPING A SAFER
3 APPENDIX 1 SAFER CARING PLAN
3 APPENDIX 1 SAFER CARING POLICY

APPENDIX 1 SAFE USE OF BED RAILS
APPENDIX 19 STANDARD BOARD OF EXAMINERS AGENDA
APPENDIX E GUIDELINES FOR MANAGERS DEALING WITH ALCOHOL

GSMNP Trichoptera barcoding samples that need to be examined

Appendix 3. Trichoptera species highlighted by barcode analysis in this study*

*Taxonomic annotations are provided when available

Category 1: Those instances where the barcode data cannot definitively identify species (taxa included in dotted lines in Figs 5, 6, 9–11).

1. Polycentropus colei Ross/P. rickeri Yamamoto (Fig. 5, both species are paraphyletic, where a P. rickeri from TN and a P. colei from NC shared barcode)

Note: Charles Parker: "PKCAD832-08 (SampleID: 07-17PKS-0738) seems to be correctly identified as Polycentropus colei. Of the three species in the group, it is closest to P. colei based on the inferior appendages. But, there are some issues. The hindwing lacks a discal cell (this group should have one), and other features of the genitalia are not terrific fits to the available illustrations."—Dec 4, 2009.

Jason Robinson: “... We've noticed some problems with the specimens we have that at first glance appear to match the published descriptions of P. colei or P. rickeri. Since these specimens are generally very rare and sequences even rarer, I am not sure that we could really add much to what Chuck has already identified other than to say ‘send us more adult males’.”—Apr 26, 2010.

Oliver Flint: “This (08OFCAD-1152) is P. rickeri, without doubt. It seems to differ from our 1 specimen of P. colei, but not profoundly. Perhaps they are synonymous, but I would want to see more P. colei examples.”—Jul 6, 2010.

Xin Zhou: “The problematic specimens showed some variations yet still followed current taxonomy. But we haven’t checked enough samples. So revision is simply not possible at the moment.”—Aug 10, 2010.

2. Cheumatopsyche campyla Ross/C. speciosa (Banks)/C. pasella Ross/C. ela Denning (Fig. 6, these 4 species are intermingled in the COI NJ tree. Some are sharing barcodes.)

Note: Members of the Cheumatopsyche campyla complex are notoriously difficult to distinguish reliably using morphology. Although a revision to Nearctic Cheumatopsyche (Gordon 1974) is widely followed by taxonomists, males with an admixture of diagnostic characters are common and the identification of both sexes remains difficult. Due to the lack of consistent diagnostic characters, a single individual is often assigned to different species if examined by more than 1 taxonomist. Indeed, such is the case for a number of specimens we show in the barcode tree (taxonomic comments can be found in the corresponding specimen records in BOLD projects). None of these conflicting identifications, however, corresponded strictly to the barcode haplotype groups (shown in Fig. 6). Possibly, the C. campyla complex has undergone recent speciation with incomplete lineage sorting that is reflected in both morphology and COI sequences, possibly accompanied with hybridization.

3. Ceraclea nepha (Ross)/C. tarsipunctata (Vorhies) (Fig. 9, two C. nepha haplotypes are placed very closely to or within C. tarsipunctata)

Note: Oliver Flint: “08OFCAD-0299, 0300, 0301 are all males and all are C. nepha, as currently understood. C. protonepha does seem to be a greater problem as it is barely distinguishable from C. nepha and I have doubts on its validity. But C. nepha and C. tarsipunctata seem quite distinct.”—Jul 6, 2010.

Roger Blahnik: “(08ONCAD-0374) color is like C. protonepha and not C. tarsipunctata.”

Jason Robinson: “(07-17PKS-0014) Specimens from this locality are distinctively aberrant from the illustrations of the type.”

Xin Zhou: “This seems to be a definite case where COI barcodes fail to distinguish two valid species.”—Aug 10, 2010.

4. Triaenodes tardus Milne/T. marginatus Sibley (Fig. 9, these 2 species are indistinguishable in the COI NJ tree)

Xin Zhou: “This seems to be a definite case where COI barcodes fail to distinguish two valid species.”—Aug 10, 2010.

5. Psilotreta rossi Wallace/P. rufa (Hagen) (Fig. 10, these 2 species are indistinguishable in the COI NJ tree)

Note: Xin Zhou: “All specimens of P. rossi and P. rufa have been re-examined and confirmed by authors of this paper. This may be another case where COI barcodes fail to distinguish relevant species. On the other hand, the NJ tree construction method may have also contributed to the current tree topology as P. rossi haplotypes may show monophyly when varied samples are analyzed (e.g., adding 1 more sequence of P. rossi and using only members of Odontoceridae as outgroups).

6. Lepidostoma pictile (Banks)/L. modestum (Banks) (Fig. 11, these 2 species are indistinguishable in the COI NJ tree)

Note: Oliver Flint: “08OFCAD-0231: male, clearly L. modestum; 08OFCAD-0232 & 0233: females that match perfectly to L. modestum but this species is very close to L. pictile in morphology."—Dec 23, 2009.

Category 2: Those instances where barcode data can identify species, but species are not monophyletic (taxa included in grey blocks with a dotted line in Figs 5, 6, 9–10).

1. Nyctiophylax affinis (Banks) (Fig. 5, two paraphyletic clusters, one of which nested within N. uncus Ross/N. banksi Morse)

Note: Roger Blahnik: “I have no reason to believe that the specimens of Nyctiophylax affinis (RBCAD-1873, RBCAD-0655, 08FLCAD-047) are misidentified. The only species they could be easily confused with is Nyctiophylax moestus and I do not think you have yet obtained sequences from that species (Author note: barcodes of N. moestus are available in this paper, which are readily distinguishable from those of both N. affinis clusters in question). As I mentioned in my previous e-mail, the species is variable, based on illustrations provided by Morse in his revision of the genus, and it is possible that the current concept of Nyctiophylax affinis includes more than one species.”—Sep 16, 2008.

2. Diplectrona modesta Banks (Fig. 6, where D. metaqui nested within a paraphyletic D. modesta, each of these two species showed deeply diverging COI haplotypes)

Note: This is an on-going project by Robinson et al. Morphological concordance in larval morphology and geographic distributions are being found for most of the COI haplotype groups revealed in this study. Some haplotypes are potentially closely related species, e.g., Diplectrona marianae Reeves.

3. Hydropsyche rossi Flint, Voshell, & Parker (Fig. 6, two clusters of H. rossi showed paraphyly, with H. simulans nested within)

Note: Roger Blahnik: “Species in this group are very hard to tell apart. Morphology suggests (08ONCAD-0393) possibly H. incommoda.”

Oliver Flint: “ (08OFCAD-0633) male, matches H. rossi most closely, but slight differences so perhaps cryptic species.”—Dec 23, 2009.

4. Ceraclea flava (Banks) (Fig. 9, two clusters of C. flava showed paraphyly, with C. ancylus nested within)

Note: Roger Blahnik: “Specimens (RBCAD-0126, RBCAD-0127) differ in size (as in most species from Florida), but not significantly in morphology. Both Minnesota and Florida specimens are very light yellowish in color (but very different in size), unlike either C. neffi or C. ancylus, which are dark brown.”—Sep 23, 2008.

5. Oecetis inconspicua (Walker) (Fig. 9, the Oecetis inconspicua complex contains at least 21 divergent COI clusters with O. nocturna nested within a paraphyletic O. inconspicua.)

Note: The likely presence of multiple species within O. inconspicua is supported by earlier observations that members of this complex show pronounced genitalic variation, even among sympatric individuals. The long-standing difficulty of species diagnosis in this group is further reflected by the fact that O. inconspicua has 8 synonyms (Morse 2010). This taxonomic uncertainty is now gaining clarity. Seven distinguishable larval types were described for this species complex (Floyd 1995). The coupling of barcode results with morphology has revealed diagnostic characters among adults of some component taxa (Zhou et al. 2010). However, extensive sampling accompanied with sequencing of type material and careful morphological examination will be required to clarify species boundaries, to properly apply the existing names and describe any new species that result.

6. Psilotreta labida Ross/P. frontalis Banks (Fig. 10, two clusters of P. frontalis nested within paraphyletic P. labida)

Note: Oliver Flint: “ (08OFCAD-0810, 08OFCAD-0811) are both male, both match P. labida”—Dec 23, 2009.

Category 3: Those instances where the barcode sequences recover monophyletic taxa, but the genetic distances are very high, possibly suggesting that current taxonomy is incomplete. Only taxa showing ≥8% intraspecific divergences are listed here. The 8% value was selected simply to highlight this issue. We are not implying a generic barcode gap value for the caddisflies examined in this study nor suggesting taxa with less than 8% divergences should not be investigated. Most of the taxa falling into this category are lacking detailed examination due to limited taxonomic investigation. Some belong to on-going projects of authors of this paper (taxa included in grey blocks with a solid line in Figs 4–7, 9–10, 12).

1. Chimarra obscura (Walker) (Fig. 4)

2. C. socia Hagen (Fig. 4)

Note: Roger Blahnik: “I note that you have 3 clades within C. socia. It is conceivable that these represent C. socia, C. moselyi, and C. parasocia, which Lago proposed as constituting a clade.”—Jan 20, 2010.

Despite the hypothesis proposed above, no investigation has been made for the North American C. socia.

3. Chimarra aterrima Hagen (Fig. 4)

4. Wormaldia moesta (Banks) (Fig. 4)

Note: Robinson has discovered males that have only one abdominal process, on VII, instead of one on both VII & VIII abdominal segments.

5. Dolophilodes distincta (Walker) (Fig. 4)

Note: Xin Zhou: “The one specimen (07GSM-0239) that is very different from the rest is a larva collected in the 2007 Bioblitz. Potentially this could be a different species, although its barcode doesn’t match any existing reference in the global library.”—Aug 10, 2010.

6. Lype diversa (Banks) (Fig. 5)

Note: Xin Zhou: “The two haplotypes that are very divergent from the main haplotype group are each presented by a single specimen from MN and FL, respectively. Examination of more specimens from these series is needed.”—Aug 10, 2010.

7. Psychomyia nomada (Ross) (Fig. 5)

Note: Xin Zhou: “The northern and southern populations of this species seem to be very different in COI barcodes. Potentially, these could be different species. Examination of more specimens from these series is needed.”—Aug 10, 2010.

8. Neureclipsis crepuscularis (Walker) (Fig. 5)

9. Polycentropus cinereus Hagen (Fig. 5)

Note: Andrew Rasmussen: “The specimen in O24 (ARPCI8) is small relative to typical P. cinereus that I have examined. This small form is fairly common on Eglin Air Force Base, but I have not seen it outside of Eglin. Possibly, it is another species in the P. cinereus Group, perhaps P. harpi or an undescribed species.”

Jason Robinson: “The Polycentropus cinereus we believe to be two species and neither is P. harpi, but P. harpi should be sequenced to clarify.”—Apr 26, 2010.

10. Parapsyche cardis Ross (Fig. 6)

Note: Roger Blahnik: “The 2 species of Parapsyche were correctly determined according to current taxonomy, but it is conceivable that there are cryptic species. There are some small differences between the specimens of P. elsis, and one specimen from our series of P. cardis (RBCAD-0016) was slightly different in color and genitalic characters. This is not any other described species.”

11. Hydropsyche morosa Hagen (Fig. 6)

Note: Xin Zhou: “The COI clustering of H. morosa is somewhat corresponding to adult H. morosa and H. bifida. But there are perhaps more than 2 species involved. I have also checked the sequenced larvae from Ontario and they also showed differences in head markings. Examination of more specimens, especially of larvae, is needed.”—Aug 10, 2010.

12. Rhyacophila glaberrima Ulmer (Fig. 7)

Note: This is an on-going project led by Robinson.

13. Rhyacophila torva Hagen (Fig. 7)

Note: Jason Robinson: “Rhyacophila torva is another mystery that demands further sampling and not much else to say about at this point.”—Apr 26, 2010.

14. Triaenodes perna Ross (Fig. 9)

Note: Xin Zhou: “T. perna and T. helo seem to be easily confused. New species belonging to this complex are being described and hopefully the taxonomic revision will help to clarify this complex. Some haplotypes examined in this study may prove to be different species.”—Aug 10, 2010.

15. Oecetis avara (Banks) (Fig. 9)

16. Molanna blenda Sibley (Fig. 10)

17. Helicopsyche borealis (Hagen) (Fig. 10)

Note: Xin Zhou: “Samples identified to H. borealis showed as least 6 distinct COI haplotype groups in our analysis. Morphological variations have been observed among and within populations. However, specimens belonging to this complex possessing some obvious morphological differences (e.g., body size, number and length of spurs on legs) do not seem to show much, if any, variation in COI (specimens collected in Mexico, not included in this paper). There are 5 synonyms that are currently recognized as H. borealis.”—Aug 10, 2010.

18. Pycnopsyche gentilis (McLachlan) (Fig. 12)

APPENDIX H SURROGATE CONSENT PROCESS ADDENDUM THE
LOCAL ENTERPRISE OFFICE CAVAN MENTORING PANEL APPENDIX
(APPENDIX) INSTRUCTIONS FOR FOREIGN EXCHANGE SETTLEMENTS OF ACCUMULATED NT

Tags: analysis in, appendix, trichoptera, highlighted, analysis, barcode, species