Library Diversity and Complementarity

Observed diversity is greatest in the plant library, where sampling has been most intensive (Table 4.6). Estimated diversity varies greatly with stringency, from over 12,000 sequences in the plant library and about 6000 sequences among fungal libraries if unique identity is defined at 90% identity (high stringency), to about 3200 plant and 4800 fungal transcripts if small differences do not split sequences into different quasispecies, i.e., at 30% identity (low stringency). The proportion of expected transcripts sequenced ( $q = S_{obs}/\hat{S}$) varies in the plant library from 7% at high stringency to 22% at low stringency, but not in the pooled fungal libraries, where q = 12% (at either high or low stringency).

Table 4.6: Observed and estimated transcript diversity in plant (Mt) and fungal (Gi) libraries. Because of the small number of samples sequenced (n) in fungal libraries, they were pooled to estimate diversity. Diversity for four identity thresholds are shown.

		IDENTITY THRESHOLD
		90%	70%	50%	30%
LIBRARY	n	$S_{obs}$
Mt Long	899	873	786	750	726
Gi Harrison	362	333	304	290	272
Gi Lammers	174	169	166	164	155
Gi Sawaki	165	164	162	161	159
Gi pooled	701	660	622	597	563
LIBRARY	ESTIMATOR	$\hat{S}$
Mt Long	ACE	12290	4587	3452	3188
	Chao 1	12505	4272	3340	3347
Gi pooled	ACE	6093	5291	5203	4776
	Chao 1	5941	5491	5130	4816

Though there is considerable overlap between plant and fungal libraries if compared in terms of hexamer composition, the plant library does not overlap with any of the three fungal libraries in terms of the composition of individual transcript quasispecies (Figure 4.7). Among the axenic fungal libraries, the Harrison and Lammers libraries are most similar, having about 97% complementarity, or 12 shared quasispecies (at 30% identity threshold). The Sawaki library is less related to the other two, sharing only eight quasispecies with the Harrison library and six with the Lammers library.

Figure 4.7: Dendrogram of libraries, clustered by complementarity of transcript quasispecies, as indicated by scale above the tree. Three libraries from Glomus intraradices (Gi Harrison, Gi Lammers, and Gi Sawaki) and one library from M. truncatula (Mt Long) are represented, using complementarity values at the 30% identity threshold as a distance criterion. Complementarity of 100% indicates that two libraries have no transcripts in common, based on this identity threshold.

Figure 4.8: Dendrogram of libraries, clustered by complementarity of transcript quasispecies, as indicated by scale above the tree. Three libraries from Glomus intraradices were pooled (Gi) and compared with axenic (KV0, MtLong, NFleaf, NFroot, NFstem) and mixed-culture (DSIR, KVnod, MHAM, and NFnod) plant libraries, using complementarity values at the 30% identity threshold as a distance criterion.

If we compare the composition of transcript quasispecies across axenic plant, axenic fungal, and mixed-culture libraries (Figure 4.8), we note the pooled fungal libraries (Gi) are clearly unrelated to plant libraries, and even unrelated to the MHAM mycorrhizal plant-root library. Among plant root libraries, the most similar pairs are KVnod and NFstem libraries, then the DSIR and MHAM libraries, joined by KV0 and NFnod, respectively. These are joined in turn by the library pair MtLong and NFleaf, and then by the NFroot library. Pure and mixed-culture plant libraries cluster together, rather than clustering into two distinct groups.